KR20050004155A - Methods for the treatment of respiratory diseases and conditions using a selective inos inhibitor - Google Patents

Abstract

The present invention relates to a method of preventing and treating a respiratory disease or condition comprising administering to a patient in need thereof an effective inhibitor of an inducible nitric oxide synthase in an amount effective for the respiratory disease or condition.

Description

METHODS FOR THE TREATMENT OF RESPIRATORY DISEASES AND CONDITIONS USING A SELECTIVE INOS INHIBITOR}

Asthma is the most common chronic disease in childhood, with about 150 million people worldwide. Due to the high prevalence of childhood asthma observed in the past decades, the prevalence of asthma is expected to increase in the near future unless appropriate precautions are taken. About 10 million Americans suffer from asthma, of which about one third are under 18. Billions of dollars a year are spent on asthma-related health products. Temporary respiratory distress, which is characteristic of asthma, has three main factors including: 1) bronchospasm, ie variable or reversible airway disorder due to airway muscle contraction, 2) inflammation inside the airway, and 3) excess airway mucus due to bronchial hyper-reactivity Caused by a combination of The triggers for asthma vary from person to person but include allergens such as dust mites and molds, environmental contaminants, viral agents, and physical activity or exercise.

Global estimates of COPD were typically based primarily on death statistics. This gives false numbers because COPD is in an undiagnosed state and is often not listed as a major cause of death or contributing factor to death. Estimation of prevalence requires the measurement of airway disorders. As a result, no country has good information based on the population on COPD prevalence. Nevertheless, these estimates show that deaths and disorders due to COPD increase over most regions for men and women. Mayo Clinic reports 85,000 deaths per year in the United States due to chronic obstructive pulmonary disease (COPD), most pneumoconiosis or chronic bronchitis. Chronic obstructive pulmonary disease refers collectively to several chronic or progressive lung diseases, including asthma bronchitis, chronic bronchitis (with normal airflow), chronic impaired bronchitis, bullous disease and pneumoconiosis, all of which involve inflammation. For example, chronic bronchitis is inflammation and possible wounds inside the bronchus that produce symptoms including chronic cough, mucus buildup, frequent flatulence and shortness of breath. Pneumoconiosis is due to a normal but chronic inflammatory response inside the airways to chronic exposure to ambient contaminants such as cigarette smoke.

Drug treatments for asthma and COPD include intravenous, oral, subcutaneous or inhaled administration of bronchodilators, including beta-adrenergic, methyl xanthine and anticholinergic agents, and mast cell modulators known as administration of corticosteroids, chromolines and tilides. -Release inhibitors, or more recently, administration of anti-leukotrienes for anti-inflammatory effects. On the other hand, the inflammatory and immune cellular and molecular mechanisms for inflammatory and immune action that play the role and progression of asthma and COPD are not well known.

Nitric oxide (NO) is a bioactive free radical gas produced by any of several isoforms of the enzyme nitric oxide synthase (NOS). The physiological activity of what was then identified as NO was first discovered in the early 1980s when it was known that vasorelaxation caused by acetylcholine was dependent on the presence of the vasculature endothelium. An endothelial derived factor (hereinafter referred to as endothelial-induced relaxation factor (EDRF)) that modulates this vasorelaxation is now known as NO produced in the vasculature endothelium by an isoform of one NOS. The activity of NO as a vasodilator has been well known for over 100 years. NO is also an active species derived from known nitrovascular dilators, including amylrite and glyceryltrinitrate. Nitric oxide is also an endogenous stimulant of soluble guanylate cyclase and thus encourages cGMP production. When NOS is inhibited by N-monomethylarginine (L-NMMA), cGMP formation is completely prevented. In addition to endothelial-dependent relaxation, NO is known to be involved in many physiological actions, including cytotoxicity of phagocytes and cell-to-cell interconnections in the central nervous system.

The identification of EDRF as NO is consistent with the discovery of biochemical pathways in which NO is synthesized from the amino acid L-arginine by the enzyme NO synthase. At least three types of NO synthase exist: (i) Ca ++ / calmodulin dependent constitutive enzymes located in the endothelium that release NO in response to receptors or physical stimuli, (ii) in response to receptors or physical stimuli Ca ++ / calmodulin dependent constitutive enzymes located in the brain that release NO, and (iii) Ca ++ induced after activation of vascular smooth muscle, macrophages, endothelial cells and many other cells by endotoxins and cytokines. Independent enzyme, 130 kD protein. Expression of this inducible nitric oxide synthase (hereafter “iNOS”) produces NO over a long period of time.

Clinical studies have shown that NO production and iNOS expression increase in several inflammatory diseases, such as rheumatoid and osteoarthritis, and are associated as a major pathological factor in these chronic inflammatory diseases.

Thus, inhibition of excessive NO production by iNOS will be anti-inflammatory. On the other hand, the production of NO from eNOS and nNOS is associated with normal physiology. Thus, all NOS inhibitors used in the treatment of inflammation are required to ensure that normal physiological control of blood pressure by eNOS-generated NO and non-adrenergic, non-cholinergic neurotransmission by nNOS-generated NO remains overall unaffected. Must be optional for iNOS.

Asthmatic patients and other patients with inflammatory airway disease have shown increased concentrations of NO in exhaled air compared to patients who are not normally affected, and exhaled NO has been proposed as a marker of airway inflammation. Increased expression of iNOS is observed in pulmonary macrophages in endothelial and bronchiectasis patients in asthma. See, eg, Barnes, PJ and Liew, FY, Immunol. Today 16 (3): 128-30 (1995).

Overproduction of NO by iNOS has been associated with the pathology of airway inflammation of asthma. See, eg, Eissa, NT, et al ., Am. J. Resp. Cell and Mol. Biol . 24 (5): 616. -20 (2001) ". In a murine model of allergic asthma, chemokines may be administered by administering one of the NOS inhibitors, L-NAME, S-ethylisothiourea, or 2-amino 5,6-dihydro 6-methyl 4H-1,3-thiazine. Airway inflammation was inhibited by downregulation of chemokine) expression. See Trifilieff, A. et al ., J. Immunol . 165 (3) 1526-33 (2000). Inhibition of iNOS with aminoguanidine has been suggested as a treatment for asthma as well as rhinitis. See Schapowal, AG and Brunnenkant, W., Allergologie 19 (1): 49 (1996). Sustained production of high levels of iNOS-generated NO is thought to be the basis for rupture of the airway endothelial, reduced ciliary function, and a balanced shift from TH-1-dominant response to TH-2-dominant response and also for eosinophils. It is thought to provide chemotactic factors, suggesting that selective inhibition of iNOS in asthma will reduce lung inflammation and improve airway function. See Manning, PT et al ., Prog. In Resp. Res . 31: 156-59 (2001).

PCT Patent Publication WO 01/05748 discloses novel oligomeric amino acid derivatives as selective iNOS inhibitors useful for the treatment of autoimmune or inflammatory diseases including asthma.

Inhibition of nuclear factor-KappaB (NF-kB) has been described for the treatment of asthma, diabetic vascular disease, heart failure and sepsis, where heparin is administered to patients to interfere with the translocation of NF-kB from the cytoplasm to the nucleus. This suppressed NF-kB expression. See PCT Publication No. 01/019376. Proteins believed to express NF-kB-dependent genes include cytokine THF, IL-1, IL-2, IL-6, IL-8, inferon-beta, inferon-gamma, tissue factor-1, complement, And iNOS.

However, cellular and molecular mechanisms based on asthma and COPD have not been known to date. In contrast to the proposal to treat such respiratory diseases by inhibiting NOS activity, it has also been suggested that the increase in the concentration of NO observed in exhaled air of other patients with asthma and lung disease is a marker of the compensatory mechanism involving NOS activity. Thus, in contrast to proposals for the treatment of asthma or COPD, including the inhibition of NOS activity, it is also suggested to dictate a method of treatment including administration of a compound that accepts, delivers or releases nitric oxide instead, or stimulates endogenous production of NO. It has been. See US Patents RE37,116 and 6,331,543.

Other studies are far from treating asthma or COPD with iNOS inhibitors. The conversion of GTP to cGMP by guanyl cyclase is thought to be stimulated by iNOS such that selective inhibition of iNOS should reduce guanylate cyclase activity and reduce the level of cGMP. US Pat. No. 6,333,354, on the other hand, teaches the treatment of acute or chronic disorders of bronchial or acute or chronic inflammation, including asthma, using a combination of PDE inhibitors and guanyl cyclase agonists. Guanyl cyclase agonists are expected to have the opposite effect of iNOS inhibitors, leading to increased guanyl cyclase activity, resulting in increased production of cGMP instead of reduced levels of cGMP.

Against this background, development of novel agents and methods for the treatment and prevention of various lung and respiratory diseases and disorders involving inflammation that may be associated with excessive iNOS activity, as well as for improved therapeutic efficacy with minimal toxicity and side effects. Increasing interest in Therefore, it would be beneficial to find and describe novel compositions and methods of treatment for the treatment and prevention of inflammation-related lung diseases and disorders.

Summary of the Invention

The present invention relates to a method of treating, preventing or inhibiting a respiratory disease or disorder in a patient in need thereof, wherein the compound of formula (I) or a pharmaceutically acceptable salt thereof, A compound of formula II or a pharmaceutically acceptable salt thereof, a compound of formula III or a pharmaceutically acceptable salt thereof, a compound of formula IV or a pharmaceutically acceptable salt thereof, a compound of formula V or a pharmaceutically acceptable salt thereof Or a pharmaceutically acceptable salt thereof, a compound of formula (VII) or a pharmaceutically acceptable salt thereof, a compound of formula (VIII) or a pharmaceutically acceptable salt thereof, Or a pharmaceutically acceptable salt thereof, and a compound of formula Is directed to a method comprising administering a pharmacologically effective allow inducible nitric oxide synthase selective inhibitors of the amount selected from the group consisting of the salts thereof, or a pharmaceutically acceptable salt or prodrug to a patient.

Where

R ¹ is selected from the group consisting of H, halo, and alkyl which may be optionally substituted with one or more halo,

R ² is selected from the group consisting of H, halo, and alkyl which may be optionally substituted with one or more halo,

At least one of R ¹ and R ² contains halo,

R ⁷ is selected from the group consisting of H and hydroxy,

J is selected from the group consisting of hydroxy, alkoxy and NR ³ R ⁴ ,

Wherein R ³ is selected from the group consisting of H, lower alkyl, lower alkylenyl and lower alkynyl,

R ⁴ is selected from the group consisting of H, and a heterocyclic ring wherein at least one of the rings is carbon and 1 to about 4 heteroatoms are independently selected from oxygen, nitrogen, and sulfur, wherein the heterocyclic ring is heteroarylamino , N-aryl-N-alkylamino, N-heteroarylamino-N-alkylamino, haloalkylthio, alkanoyloxy, alkoxy, heteroaralkoxy, cycloalkoxy, cycloalkenyloxy, hydroxy, amino, thio, Nitro, lower alkylamino, alkylthio, alkylthioalkyl, arylamino, aralkylamino, arylthio, alkylsulfinyl, alkylsulfonyl, alkylsulfonamido, alkylaminosulfonyl, amidosulfonyl, monoalkyl amidosulfonyl , Dialkyl amidosulfonyl, monoarylamidosulfonyl, arylsulfonamido, diarylamidosulfonyl, monoalkyl monoaryl amidosulfonyl, arylsulfinyl, arylsulfonyl, Teraroarylthio, heteroarylsulfinyl, heteroarylsulfonyl, alkanoyl, alkenoyl, aroyl, heteroaroyl, aralkanoyl, heteroarkanoyl, haloalkanoyl, alkyl, alkenyl, alkynyl, alkylene Dioxy, haloalkylenedioxy, cycloalkyl, cycloalkenyl, lower cycloalkylalkyl, lower cycloalkenylalkyl, halo, haloalkyl, haloalkoxy, hydroxyhaloalkyl, hydroxyaralkyl, hydroxyalkyl, Hydroxyheteroaralkyl, haloalkoxyalkyl, aryl, aralkyl, aryloxy, aralkoxy, aryloxyalkyl, saturated heterocyclyl, partially saturated heterocyclyl, heteroaryl, heteroaryloxy, heteroaryloxyalkyl, arylalkyl , Heteroarylalkyl, arylalkenyl, heteroarylalkenyl, cyanoalkyl, dicyanoalkyl, carboxamidoalkyl, dicarboxamidoalkyl, cyanocarboalkoxy Kil, carboalkoxyalkyl, dicaralkoxyalkyl, cyanocycloalkyl, dicyanocycloalkyl, carboxamidocycloalkyl, dicarboxamidocycloalkyl, carboalkoxycyanocycloalkyl, carboalkoxycycloalkyl, di Carboalkoxycycloalkyl, formylalkyl, acylalkyl, dialkoxyphosphonoalkyl, dialkoxyphosphonoalkyl, phosphonoalkyl, dialkoxyphosphonoalkoxy, dialkoxyphosphonoalkoxy, phosphonoalkoxy, dialkoxyphosphonoalkyl Optionally substituted with amino, dialkoxyphosphonoalkylamino, phosphonoalkylamino, dialkoxyphosphonoalkyl, dialkylalkylphosphonoalkyl, guanidino, amidino or acylamino.

Where

X is selected from the group consisting of -S-, -S (O)-and -S (O) ₂ , preferably X is -S-,

R ¹² is C ₁ -C ₆ alkyl, C ₂ -C ₆ alkenyl, C ₂ -C ₆ alkynyl, C ₁ -C ₅ alkoxy-C ₁ alkyl and C ₁ -C ₅ alkylthio-C ₁ alkyl, wherein Each of these groups is optionally substituted with one or more substituents selected from the group consisting of -OH-, alkoxy and halogen), preferably R ¹² consists of -OH, alkoxy and halogen C ₁ -C ₆ alkyl optionally substituted with a substituent selected from the group

As for R ¹³ and R ¹⁸ ,

R ¹⁸ is selected from the group consisting of —OR ²⁴ and —N (R ²⁵ ) (R ²⁶ ), and R ¹³ is —H, —OH, —C (O) —R ²⁷ , —C (O) —OR ²⁸ And -C (O) -SR ²⁹ , or

R ¹⁸ is -N (R ³⁰ )-, R ¹³ is -C (O)-, and R ¹⁸ and R ¹³ together with the atoms to which they are attached form a ring, or

R ¹⁸ is -O-, R ¹³ is -C (R ³¹ ) (R ³² )-, wherein R ¹⁸ and R ¹³ together with the atoms attached thereto form a ring,

When R ¹³ is -C (R ³¹ ) (R ³² )-, R ¹⁴ is -C (O) -OR ³³ , otherwise R ¹⁴ is -H,

R ¹¹ , R ¹⁵ , R ¹⁶ and R ¹⁷ are independently —H, halogen, C ₁ -C ₆ alkyl, C ₂ -C ₆ alkenyl, C ₂ -C ₆ alkynyl and C ₁ -C ₅ alkoxy- Selected from the group consisting of C ₁ alkyl,

R ¹⁹ and R ²⁰ are independently selected from the group consisting of -H, C ₁ -C ₆ alkyl, C ₂ -C ₆ alkenyl, C ₂ -C ₆ alkynyl and C ₁ -C ₅ alkoxy-C ₁ alkyl ,

As for R ²¹ and R ²² ,

R ²¹ is selected from the group consisting of -H, -OH, -C (O) -OR ³⁴ and -C (O) -SR ³⁵ , and R ²² is -H, -OH, -C (O) -OR ³⁶ And -C (O) -SR ³⁷ , or

R ²¹ is -O-, R ²² is -C (O)-, and R ²¹ and R ²² together with the atoms to which they are attached form a ring, or

R ²¹ is -C (O)-, R ²² is -O-, and R ²¹ and R ²² together with the atoms attached to them form a ring,

R ²³ is C ₁ alkyl,

R ²⁴ is selected from the group consisting of —H and C ₁ -C ₆ alkyl, and when R ²⁴ is C ₁ -C ₆ alkyl, R ²⁴ is selected from the group consisting of cycloalkyl, heterocyclyl, aryl and heteroaryl Optionally substituted with one or more residues,

As for R ²⁵ and R ²⁶ ,

R ²⁵ is selected from the group consisting of -H, alkyl and alkoxy, R ²⁶ is -H, -OH, alkyl, alkoxy, -C (O) -R ³⁸ , -C (O) -OR ³⁹ and -C ( O) -SR ⁴⁰ , wherein when R ²⁵ and R ²⁶ are independently alkyl or alkoxy, R ²⁵ and R ²⁶ are independently selected from the group consisting of cycloalkyl, heterocyclyl, aryl and heteroaryl Optionally substituted with one or more residues, or

R ²⁵ is -H, R ²⁶ is selected from the group consisting of cycloalkyl, heterocyclyl, aryl and heteroaryl,

R ²⁷ , R ²⁸ , R ²⁹ , R ³⁰ , R ³¹ , R ³² , R ³³ , R ³⁴ , R ³⁵ , R ³⁶ , R ³⁷ , R ³⁸ , R ³⁹ and R ⁴⁰ are independently composed of -H and alkyl Selected from the group wherein the alkyl is optionally substituted with one or more residues selected from the group consisting of cycloalkyl, heterocyclyl, aryl and heteroaryl,

Any R ¹¹ , R ¹² , R ¹³ , R ¹⁴ , R ¹⁵ , R ¹⁶ , R ¹⁷ , R ¹⁸ , R ¹⁹ , R ²⁰ , R ²¹ , R ²² , R ²³ , R ²⁴ , R ²⁵ , R ²⁶ , R ²⁷ , R ²⁸ , R ²⁹ , R ³⁰ , R ³¹ , R ³² , R ³³ , R ³⁴ , R ³⁵ , R ³⁶ , R ³⁷ , R ³⁸ , R ³⁹ and R ⁴⁰ are independently alkyl, alkenyl, alkyne When the residue is selected from the group consisting of one, alkoxy, alkylthio, cycloalkyl, heterocyclyl, aryl and heteroaryl, the residue is optionally substituted with one or more substituents selected from the group consisting of -OH, alkoxy and halogen .

Where

R ⁴¹ is H or methyl and R ⁴² is H or methyl.

Where

R ⁴³ is hydrogen, halo, C ₁ -C ₅ alkyl, and C ₁ -C ₅ alkyl substituted with alkoxy or one or more halo,

R ⁴⁴ is hydrogen, halo, C ₁ -C ₅ alkyl, and C ₁ -C ₅ alkyl substituted with alkoxy or one or more halo,

R ⁴⁵ is C ₁ -C ₅ alkyl, or C ₁ -C ₅ alkyl substituted with alkoxy or one or more halo.

Wherein R ⁴⁶ is C ₁ -C ₅ alkyl wherein said C ₁ -C ₅ alkyl is optionally substituted with halo or alkoxy and said alkoxy is optionally substituted with one or more halo.

Where

R ⁴⁷ is hydrogen, halo, C ₁ -C ₅ alkyl, and C ₁ -C ₅ alkyl substituted with alkoxy or one or more halo,

R ⁴⁸ is hydrogen, halo, C ₁ -C ₅ alkyl, and C ₁ -C ₅ alkyl substituted with alkoxy or one or more halo,

R ⁴⁹ is C ₁ -C ₅ alkyl, or C ₁ -C ₅ alkyl substituted with alkoxy or one or more halo.

Where

R ⁵⁰ is C ₁ -C ₅ alkyl, wherein the C ₁ -C ₅ alkyl is optionally substituted with halo or alkoxy and the alkoxy is optionally substituted with one or more halo.

Where

R ⁵⁰ is selected from the group consisting of hydrogen, halo and C ₁ -C ₅ alkyl, wherein said C ₁ -C ₅ alkyl is optionally substituted with halo or alkoxy, said alkoxy is optionally substituted with one or more halo,

R ⁵¹ is selected from the group consisting of hydrogen, halo and C ₁ -C ₅ alkyl, wherein said C ₁ -C ₅ alkyl is optionally substituted with halo or alkoxy, said alkoxy is optionally substituted with one or more halo,

R ⁵² is C ₁ -C ₅ alkyl, wherein the C ₁ -C ₅ alkyl is optionally substituted with halo or alkoxy, the alkoxy is optionally substituted with one or more halo,

R ⁵³ is selected from the group consisting of hydrogen, halo and C ₁ -C ₅ alkyl, wherein said C ₁ -C ₅ alkyl is optionally substituted with halo or alkoxy, said alkoxy is optionally substituted with one or more halo,

R ⁵⁴ is selected from the group consisting of halo and C ₁ -C ₅ alkyl, wherein the C ₁ -C ₅ alkyl is optionally substituted with halo or alkoxy and the alkoxy is optionally substituted with one or more halo.

Where

R ⁵⁵ is C ₁ -C ₅ alkyl, wherein the C ₁ -C ₅ alkyl is optionally substituted with halo or alkoxy and the alkoxy is optionally substituted with one or more halo.

In other exemplary compounds, the inducible nitric oxide synthase selective inhibitor is a compound of Formula (XI) or a pharmaceutically acceptable salt thereof. Compound (XI) has already been described in International Publication No. WO 00/26195, published May 11, 2000, which is incorporated herein by reference.

2S-amino-6-[(1-iminooethyl) amino] -N- (1H-tetrazol-5-yl) hexaneamide, hydrate, dihydrochloride

The present invention also contemplates the use of other selective iNOS inhibitors. By way of example, iNOS selective inhibitors also useful in the present invention are described in US Pat. No. 6,355,689 to Beswick et al., Filed November 29, 2000 and issued March 12, 2002, which is represented by Formula XII: Selective iNOS inhibitors are described and claimed.

Where

R ⁷⁹ is selected from C _1-4 alkyl, C _3-4 cycloalkyl, C _1-4 hydroxyalkyl and C _1-4 haloalkyl.

The description of US Pat. No. 6,355,689 states that R ⁷⁹ is preferably C _1-4 alkyl, most preferably methyl. Particular embodiments and compositions disclosed in US Pat. No. 6,355,689 and useful for use in the methods of the present invention include:

S-((R) -2- (1-iminoethylamino) propyl) -L-cysteine,

S-((S) -2- (1-iminoethylamino) propyl) -L-cysteine,

S-((R / S) -2- (1-iminoethylamino) propyl) -L-cysteine,

S-((R) -2- (1-iminoethylamino) propyl) -D-cysteine,

S-((S) -2- (1-iminoethylamino) propyl) -D-cysteine,

S-((R / S) -2- (1-iminoethylamino) propyl) -D-cysteine,

S-((R / S) -2- (1-iminoethylamino) butyl) -L-cysteine,

S-((R / S) -2- (1-iminoethylamino, 2-cyclopropyl) ethyl) -L-cysteine and

S-((R / S) -2- (1-iminoethylamino, 3-hydroxy) propyl) -L-cysteine,

Or pharmaceutically acceptable salts, solvates or physiologically active derivatives thereof.

Such selective iNOS inhibitors are believed to act by competing with arginine as a substrate for iNOS enzymes. Another measure for the inhibition of iNOS is International Patent Application No. PCT / US98 / 03176, published on August 27, 1998, by Arnaiz et al., Publication No. WO 98/37079 (Berlex Laboratories, Inc.). Richmond, CA 94804-0099 and Pharmacopeia, Inc. Princeton, NJ 08540). The Arnaise application describes inhibitors of iNOS monomer dimerization. The iNOS enzyme is a homodimer and each monomer has a reductase domain to incorporate binding sites for flavin cofactors (FAD and FMN) and NADPH. Reductase domains supply electrons to the oxidase domain of other monomers, where L-arginine is oxidized at the active site, which incorporates the heme group (Fe) cytochrome P-450 domain. Tetrahydrobiopterin (BH4) is required for homodimerization and regulates the heme redox state during electron transfer. iNOS monomers are inert and dimerization is required for activity.

Accordingly, in another embodiment of the present invention, the selective iNOS inhibitor is a dimerization inhibitor represented by the compound of formula (XIII), (XIV) or (XV) as a single stereoisomer or mixture thereof, or a pharmaceutically acceptable salt thereof. .

In the above formulas,

A is -R ⁵⁶ , -OR ⁵⁶ , C (O) N (R ⁵⁶ ) R ⁵⁷ , P (O) [N (R ⁵⁶ ) R ⁵⁷ ] ₂ , -N (R ⁵⁶ ) C (O) R ⁵⁷ , -N (R ⁷⁶ ) C (O) OR ⁵⁶ , -N (R ⁵⁶ ) R ⁷⁶ , -N (R ⁷¹ ) C (O) N (R ⁵⁶ ) R ⁷¹ , -S (O) _t R ⁵⁶ ,- SO ₂ NHC (O) R ⁵⁶ , -NHSO ₂ R ⁷⁷ , -SO ₂ NH (R ⁵⁶ ) H, -C (O) NHSO ₂ R ⁷⁷ and -CH = NOR ⁵⁶ ,

Each X, Y and Z is independently N or C (R ¹⁹ ),

Each U is N or C (R ⁶⁰ ), where U is only N when X is N and Z and Y are CR ⁷⁴ ,

V is N (R ⁵⁹ ), S, O or C (R ⁵⁹ ) H,

Each W is N or CH,

Q is selected from the group consisting of a direct bond, -C (O)-, -O-, -C (= NR ⁵⁶ )-, S (O) _t and -N (R ⁶¹ )-,

m is 0 or an integer from 1 to 4,

n is 0 or an integer of 1 to 3,

q is 0 or 1,

r is 0 or 1, but when Q and V are heteroatoms, m, q and r cannot all be 0,

A is -OR ⁵⁶ , N (R ⁵⁶ ) C (O) R ⁵⁷ , -N (R ⁷¹ ) C (O) OR ⁵⁷ , -N (R ⁵⁶ ) R ⁷⁶ , -N (R ⁷¹ ) C (O) When N (R ⁵⁶ ) R ⁷¹ , -S (O) _t R ⁵⁶ , where t is 0, or -NHSO ₂ R ⁷⁷ , all of n, q and r cannot be 0, and Q is hetero A and A are -OR ⁵⁶ , N (R ⁵⁶ ) C (O) R ⁵⁷ , -N (R ⁷¹ ) C (O) OR ⁵⁷ , -N (R ⁵⁶ ) R ⁷⁶ , N (R ⁷¹ ) C (O ) N (R ⁵⁶ ) R ⁷¹ , -S (O) _t R ⁵⁶ (where t is 0) or -NHSO ₂ R ⁷⁷ , m and n cannot both be 0,

t is 0, 1 or 2,

Is optionally substituted N-heterocyclyl,

Is optionally substituted carbocyclyl or optionally substituted N-heterocyclyl,

Each of R ⁵⁶ and R ⁵⁷ is hydrogen, optionally substituted C ₁ -C ₂₀ alkyl, optionally substituted cycloalkyl, — [C ₀ -C ₈ alkyl] -R ⁶⁴ ,-[C ₂ -C ₈ alkenyl ] -R ⁶⁴ ,-[C ₂ -C ₈ alkynyl] -R ⁶⁴ ,-[C ₂ -C ₈ alkyl] -R ⁶⁵ (optionally substituted with hydroxy),-[C ₁ -C ₈ ]- R ⁶⁶ (optionally substituted with hydroxy), independently selected from the group consisting of optionally substituted heterocyclyl, or

R ⁵⁶ and R ⁵⁷ are N-heterocyclyl optionally substituted with a nitrogen atom attached to them,

R ⁵⁸ is hydrogen, alkyl, cycloalkyl, optionally substituted aryl, haloalkyl,-[C ₁ -C ₈ alkyl] -C (O) N (R ⁵⁶ ) R ⁵⁷ ,-[C ₁ -C ₈ alkyl] -N (R ⁵⁶ ) R ⁵⁷ ,-[C ₁ -C ₈ alkyl] -R ⁶³ ,-[C ₂ -C ₈ alkyl] -R ⁶⁵ ,-[C ₁ -C ₈ alkyl] -R ⁶⁶ , and hetero or a direct bond with R ⁵⁸ - heterocyclyl selected from the group consisting of (halo, alkyl, alkoxy and imidazole optionally substituted with one or more substituents selected from the group consisting of thiazolyl) or, or Q is -N (R ⁵⁸⁾ R ⁵⁸ may further be aminocarbonyl, alkoxycarbonyl, alkylsulfonyl, monoalkylaminocarbonyl, dialkylaminocarbonyl and -C (= NR ⁷³ ) -NH ₂ , or -QR ⁵⁸ Together with -C (O) OH, -C (O) N (R ⁵⁶ ) R ⁵⁷ or Indicates,

R ⁵⁹ is selected from the group consisting of hydrogen, alkyl, aryl, aralkyl and cycloalkyl,

When A is —R ⁵⁶ or —OR ⁵⁶ , R ⁵⁹ may not be hydrogen; when V is CH, R ⁵⁹ may additionally be hydroxy,

R ⁶⁰ is hydrogen, alkyl, aryl, aralkyl, haloalkyl, optionally substituted aralkyl, optionally substituted aryl, -OR ⁷¹ , -S (O) _t -R ⁷¹ , N (R ⁷¹ ) R ⁷⁶ , N (R ⁷¹ ) C (O) N (R ⁵⁶ ) R ⁷¹ , N (R ⁷¹ ) C (O) OR ⁷¹ , N (R ⁷¹ ) C (O) R ⁷¹ ,-[C ₀ -C ₈ alkyl] -C (H) [C (O) R ⁷¹ ] ₂ and-[C ₀ -C ₈ alkyl] -C (O) N (R ⁵⁶ ) R ⁷¹ ,

R ⁶¹ is hydrogen, alkyl, cycloalkyl,-[C ₁ -C ₈ alkyl] -R ⁶³ ,-[C ₂ -C ₈ ] alkyl] -R ⁶⁵ ,-[C ₁ -C ₈ alkyl] -R ⁶⁶ , Acyl, -C (O) R ⁶³ , -C (O)--[C ₁ -C ₈ alkyl] -R ⁶³ , alkoxycarbonyl, optionally substituted aryloxycarbonyl, optionally substituted aralkoxycarbonyl , Alkylsulfonyl, optionally substituted aryl, optionally substituted heterocyclyl, alkoxycarbonylalkyl, carboxyalkyl, optionally substituted arylsulfonyl, aminocarbonyl, monoalkylaminocarbonyl, dialkylaminocarbonyl , Optionally substituted arylaminocarbonyl, aminosulfonyl, monoalkylaminosulfonyl dialkylaminosulfonyl, arylaminosulfonyl, arylsulfonylaminocarbonyl, optionally substituted N-heterocyclyl, -C ( = NH) -N (CN) R ⁵⁶ , -C (O) R ⁷⁸ -N (R ⁵⁶ ) R ⁵⁷ , -C (O) -N (R ⁵⁶ ) R ⁷⁸ -C (O) OR ⁵⁶ Selected from

Each of R ⁶³ and R ⁶⁴ is optionally substituted with one or more substituents selected from the group consisting of haloalkyl, cycloalkyl (optionally substituted with halo, cyano, alkyl or alkoxy), carbocyclyl (halo, alkyl and alkoxy) And heterocyclyl (optionally substituted with alkyl, aralkyl or alkoxy), and

Each R ⁶⁵ is halo, alkoxy, optionally substituted aryloxy, optionally substituted aralkoxy, optionally substituted -S (O) _t -R ⁷⁷ , acylamino, amino, monoalkylamino, dialkylamino, Independently selected from the group consisting of (triphenylmethyl) amino, hydroxy, mercapto, alkylsulfonamido,

Each R ⁶⁶ is independently selected from the group consisting of cyano, di (alkoxy) alkyl, carboxy, alkoxycarbonyl, aminocarbonyl, monoalkylaminocarbonyl and dialkylaminocarbonyl,

Each of R ⁶⁷ , R ⁶⁸ , R ⁶⁹ , R ⁷⁰ , R ⁷² and R ⁷⁵ is independently hydrogen or alkyl,

Each R ⁷¹ is independently hydrogen, alkyl, optionally substituted aryl, optionally substituted aralkyl or cycloalkyl,

R ⁷³ is hydrogen, NO ₂ or toluenesulfonyl,

Each R ⁷⁴ is independently hydrogen, alkyl (optionally substituted with hydroxy), cyclopropyl, halo or haloalkyl,

Each R ⁷⁶ is independently hydrogen, alkyl, cycloalkyl, optionally substituted aryl, optionally substituted aralkyl, —C (O) R ⁷⁷ or —SO ₂ R ⁷⁷ , or

R ⁷⁶ is N-heterocyclyl optionally substituted with R ⁵⁶ and the nitrogen attached thereto; or

R ⁷⁶ is N-heterocyclyl optionally substituted with R ⁷¹ and nitrogen attached thereto,

Each R ⁷⁷ is independently alkyl, cycloalkyl, optionally substituted aryl or optionally substituted aralkyl,

R ⁷⁸ is an amino acid residue.

Another iNOS dimerization inhibitor, 3- (2,4-difluorophenyl) -6- {2- [4- (1H-imidazol-1-ylmethyl) phenoxy] ethoxy} -2-phenylpyridine (PPA250) is described in Otsusuka et al., J. Phamacol Exp Ther Vol. 303, Issue 1, 52-57, October 2002. PPA250 has the following structure.

Thus, in another embodiment of the present invention, compound PPA250 may be used as a selective iNOS inhibitor.

Respiratory diseases or disorders to be treated include, for example, allergen-induced asthma, exercise-induced asthma, contaminant-induced asthma, asthma diseases including cold-induced asthma, virus-induced asthma, or chronic airflow with normal airflow. There are bronchitis, chronic obstructive pulmonary disease, including chronic bronchitis with airway disorders, pneumonia, asthma bronchitis, and bullous disease as well as cystic fibrosis. In addition, respiratory diseases or diseases include pigeon fancier's disease, farmer's lung, acute respiratory distress syndrome, pneumonia, aspiration or inhalation injury, fatty embolism in the lung, acidosis inflammation of the lung, acute lung edema, acute altitude sickness, After cardiac surgery, any of a wide range of respiratory diseases or diseases including acute pulmonary hypertension, permanent pulmonary hypertension in newborns, prenatal respiratory syndrome, vitreous disease, acute pulmonary embolism, heparin-protamine reaction, sepsis, persistent asthma and hypoxia Can be.

The present invention generally relates to medical treatment methods using selective inhibitors of inducible forms of nitric oxide synthase (iNOS), more specifically respiratory organs including lung disease collectively referred to as asthma disease as well as chronic obstructive pulmonary disease (COPD). A novel method useful for the medical prevention and treatment of diseases and conditions.

1 is a graph of medium nitrate content after incubating human primary airway endothelial cells in the presence or absence of L-NIL for 24 hours in the presence of 50ng / ml 1L-1β, TNF-α and IFN-γ (cyt).

FIG. 2 shows the results of isolation of cellular protein 3-8% tris-acetate polyacrylamide gel and immunoblot against iNOS protein.

FIG. 3 shows (A) 20 mg of iNOS selective inhibitor (Compound NN) in mild to moderate asthmatic patients (▲) compared to placebo (Δ) and healthy patients (●) compared to placebo (○) and (B) ) Changes in nitric oxide (NO) levels of exhalation after oral administration of compound NN 200 mg.

4 depicts the effect of oral administration of compound NN on FEV ₁ , blood pressure and heart rate.

The following detailed description is provided to assist those skilled in the art to practice the present invention. On the other hand, the following detailed description should not be considered as limiting the invention unnecessarily, since modifications and variations are made in the exemplary embodiments discussed herein without departing from the scope of the appended claims by those skilled in the art.

The contents of each of the main references cited herein, including the contents of the references cited in the main references, are incorporated herein by reference in their entirety.

The present invention includes a method of treatment using a novel selective iNOS inhibitor for treating or preventing a respiratory disease or disorder, including a method of treatment using a medicament for the prevention and treatment of a respiratory disease or disorder, the respiratory disease or disorder Examples include asthma, including allergen-induced asthma, exercise-induced asthma, contaminant-induced asthma, cold-induced asthma, virus-induced asthma, or chronic bronchitis with normal airflow, chronic bronchitis with airway disorders. Chronic obstructive pulmonary disease, including chronic chronic bronchitis, pneumonia, bronchitis, and bullous disease, and cystic fibrosis, pigeon breeder's disease, farmer's lung, acute respiratory distress syndrome, pneumonia, aspiration or inhalation injury, in the lungs Fatty embolism, acidosis inflammation of the lungs, acute pulmonary edema, acute altitude sickness, after cardiac surgery, acute pulmonary hypertension, permanent pulmonary hypertension in newborns, Post-natal respiratory syndrome, vitreous membrane disease, acute pulmonary thromboembolism, heparin-protamine response, sepsis, asthma persistence and other inflammatory lung diseases including hypoxia.

The method of treatment comprises administering to the patient an optional inhibitor of inducible nitric oxide synthase having a formula selected from Formulas I-XI in an amount effective for respiratory disease.

a. Justice

The terms “nitric oxide synthase” and “NOS” as used interchangeably herein refer to any of the isoforms of the enzymatic nitric oxide synthase, including eNOS, nNOS and iNOS.

As used herein interchangeably, the terms "inducible nitric oxide synthase", "NOS-2" and "iNOS" refer to the Ca ⁺² -independent inducible isoform of the enzyme nitric oxide synthase.

"Nitric oxide synthase inhibitor" and "NOS inhibitor" refer to compounds that reduce the physiological effects of nitric oxide synthase enzymes. Such inhibitors may be selective for a particular isoform of nitric oxide synthase or may be substantially non-selective, ie highly effective for two or more isoforms of nitric oxide synthase.

The terms "selective nitric oxide synthase inhibitor" and "selective NOS inhibitor" preferably refer to a compound capable of reducing the pharmacological effect of certain isoforms of nitric oxide synthase compared to other isoforms of nitric oxide synthase. do.

The terms "selective inducible nitric oxide synthase inhibitor", "selective NOS-2 inhibitor" and "selective iNOS inhibitor" used interchangeably are preferably nitric oxide synthase compared to other isoforms of nitric oxide synthase. It means a compound capable of reducing the pharmacological effect of the calcium ion-independent isoform of. In one embodiment, selective inhibition of iNOS is shown relative to endogenous NOS or neuronal NOS such that selective iNOS inhibitors exhibit an efficacy of less than 100 mg / kg in vivo (ED ₅₀ ). In other embodiments, the selective iNOS inhibitor shows selective inhibition of iNOS over endogenous NOS or neuronal NOS, exhibiting an efficacy of less than 10 mg / kg (ED ₅₀ ) in rodent animal endotoxin models. In a further embodiment, about 20-fold selectivity for eNOS is measured when measured by an increase in mean arterial blood pressure. In another embodiment, the iNOS inhibitor exhibits at least about 100-fold selectivity to eNOS as measured by an increase in mean arterial blood pressure. In another embodiment, the iNOS inhibitor exhibits about 20-fold selectivity for nNOS as measured by gastrointestinal transit or decreased erection of the penis. In another embodiment, the iNOS inhibitor exhibits at least about 100-fold selectivity to nNOS as measured by gastrointestinal transit or decreased erection of the penis.

The term "alkyl" alone or in combination means a linear or branched acyclic alkyl radical containing preferably 1 to about 10 carbon atoms, more preferably 1 to about 6 carbon atoms. "Alkyl" also includes cyclic alkyl radicals containing 3 to about 7 carbon atoms, preferably 3 to 5 carbon atoms. The alkyl radicals may be optionally substituted with groups defined below. Examples of such radicals are methyl, ethyl, chloroethyl, hydroxyethyl, n-propyl, isopropyl, n-butyl, cyanobutyl, isobutyl, s-butyl, t-butyl, pentyl, aminopentyl, iso- Amyl, hexyl, octyl and the like.

The term "alkenyl" refers to a linear or branched acyclic hydrocarbon radical having one or more double bonds, which radicals are from 2 to about 6 carbon atoms, preferably 2 to about 4 carbon atoms, more preferably Contains 2 to about 3 carbon atoms. The alkenyl radicals may be optionally substituted with groups defined below. Examples of suitable alkenyl radicals include propenyl, 2-chloropropyleneyl, buten-1-yl, isobutenyl, penten-1-yl, 2-methylbuten-1-yl, 3-methylbuten-1-yl, Hexen-1-yl, 3-hydroxyhexen-1-yl, hepten-1-yl, octen-1-yl and the like.

The term "alkynyl" refers to a linear or branched unsaturated acyclic hydrocarbon having one or more triple bonds, which radicals are from 2 to about 6 carbon atoms, preferably 2 to about 4 carbon atoms, more preferably Contains 2 to about 3 carbon atoms. The alkynyl radical may be optionally substituted with a group defined below. Examples of suitable alkynyl radicals are ethynyl, propynyl, hydroxypropynyl, butyn-1-yl, butyn-2-yl, pentin-1-yl, pentyn-2-yl, 4-methoxypentin-2- 1, 3-methylbutyn-1-yl, hexyn-1-yl, hexyn-2-yl, hexyn-3-yl, 3,3-dimethylbutyn-1-yl radicals and the like.

The term "alkoxy" is a linear or branched oxy-containing radical, each having an alkyl moiety of 1 to about 6 carbon atoms, preferably 1 to about 3 carbon atoms, including methoxy radicals. The term "alkoxyalkynyl" also includes alkyl radicals having one or more alkoxy radicals attached to the alkyl radical, i.e., forming monoalkoxyalkyl and dialkoxyalkyl radicals. Examples of such radicals include methoxy, ethoxy, propoxy, butoxy and t-butoxy alkyl. "Alkoxy" radicals may be further substituted with one or more halo atoms such as fluoro, chloro or bromo to form "haloalkoxy" radicals. Examples of such radicals are fluoromethoxy, chloromethoxy, trifluoromethoxy, difluoromethoxy, trifluoroethoxy, fluoroethoxy, tetrafluoroethoxy, pentafluoroethoxy and fluoropropoxy Can be mentioned.

The term "alkylthio" includes radicals attached to divalent sulfur atoms and containing linear or branched alkyl radicals of 1 to about 6 carbon atoms. An example of "lower alkylthio" is methylthio (CH ₃ -S-).

The term "alkylthioalkyl" includes alkylthio radicals attached to an alkyl group. Examples of such radicals include methylthiomethyl.

The term "halo" means halogen such as fluorine, chlorine, bromine or iodine atoms.

The term "heterocyclyl" refers to a saturated or unsaturated monocyclic or polycyclic carbocycle in which one or more carbon atoms are replaced by N, S, P or O. This includes, for example, the following structure.

In the above formulas,

Z, Z ¹ , Z ² or Z ³ is C, S, P, O or N, wherein one of Z, Z ¹ , Z ² or Z ³ is other than carbon, but is attached to another Z atom by a double bond or Or is not O or S when attached to another O or S atom. In addition, it is understood that the optional substituents are attached to Z, Z ¹ , Z ² or Z ³ only when each is C.

The term “heterocyclyl” also refers to fully saturated ring structures such as piperazinyl, dioxanyl, tetrahydrofuranyl, oxiranyl, aziridinyl, morpholinyl, pyrrolidinyl, piperidinyl, thiazolidinyl and It includes others. The term "heterocyclyl" also includes partially unsaturated ring structures such as dihydrofuranyl, pyrazolinyl, imidazolinyl, pyrrolidinyl, chromanyl, dihydrothiophenyl and the like.

The term "heteroaryl" refers to a fully unsaturated heterocycle.

In "heterocycle" or "heteroaryl", the point of attachment to the molecule may be present at the hetero atom or elsewhere in the ring.

The term "cycloalkyl" refers to a monocyclic or polycyclic carbocycle, each ring containing 3 to about 7 carbon atoms, preferably 3 to about 5 carbon atoms. Examples include radicals such as cyclopropyl, cyclobutyl, cyclopentyl, cyclohexyl, cycloalkenyl and cycloheptyl. The term "cycloalkyl" further includes a spiro system in which the cycloalkyl ring has carbon ring atoms in common with the seven-membered heterocyclic ring of benzothiefine.

The term "oxo" means double bonded oxygen.

The term "alkoxy" means a radical comprising an alkyl radical bonded to an oxygen atom, such as a methoxy radical. More preferred alkoxy radicals are "lower alkoxy" radicals having 1 to about 10 carbon atoms. Even more preferred alkoxy radicals have 2 to about 6 carbon atoms. Examples of such radicals include methoxy, ethoxy, propoxy, isopropoxy, butoxy and t-butoxy.

The term "aryl" refers to fully unsaturated monocyclic or polycyclic carbocycles, including but not limited to saturated or unsaturated phenyl, naphthyl or anthracenyl.

The phrase "optionally substituted" means that the radicals indicated may be substituted with hydrogen but are not essential. As such, the phrase “optionally substituted with one or more” means that one or more substitutions are also considered when a substitution is made at the indicated residues. In this regard, when one or more optional substituents are present, the substitution may be optional or a combination of substituents may be selected, or one or more identical substituents may be selected. By way of non-limiting example, the phrase “C ₁ -C ₅ alkyl optionally substituted with one or more halo or alkoxy” refers to, for example, methyl, ethyl, propyl, butyl or pentyl at all substitutable positions with hydrogen, fluorine, chlorine or other It should be meant to have halogen, methoxy, ethoxy, propoxy, isobutoxy, t-butoxy, pentoxy or other alkoxy radicals, and combinations thereof. Non-limiting examples include propyl, i-propyl, methoxypropyl, fluoromethyl, fluoropropyl, 1-fluoro-methoxymethyl and the like.

Where a compound is described by both a structure and a name, the name is intended to correspond to the structure mentioned and similarly the structure is intended to correspond to the name mentioned.

As used herein, the term “patient” refers to an animal, in one embodiment a mammal, and in one exemplary embodiment, in particular, a human being for the treatment, observation or experiment.

As used herein, the terms “administration” and “treatment” refer to all treatments, actions, applications, cures, etc., wherein the patient, in particular a human being, receives medical assistance for the purpose of improving the patient's disease directly or indirectly. Refers to.

The term "therapeutic compound" as used herein refers to a compound useful for the prevention or treatment of a respiratory disease or condition.

As used herein, the term "therapeutically effective" refers to a specific amount of therapeutic compound, or a combination of specific amounts of therapeutic compound in a combination therapy. Such amounts or combined amounts serve the purpose of preventing, avoiding, alleviating or eliminating a respiratory disease or condition.

The term “prodrug” refers to a compound that is a drug precursor where subsequent administration and subsequent uptake into the patient is converted to the active species through any process, such as metabolic, in vivo. Other products from the conversion process are easily disposed of in the body. More preferred prodrugs involve a conversion process that produces a product that is generally accepted as safe.

The term "asthma" is one of three main factors, including 1) bronchospasm, a variable and reversible airway disorder due to airway muscle contraction, 2) inflammation inside the airway, and 3) excess mucus in the airway due to bronchial hyper-response Refers to respiratory disorders characterized by temporary respiratory distress caused by one or a combination thereof, exposure to allergens or combinations of allergens such as dust mites and fungi, viral or bacterial infections, especially infections caused by “cold” viruses, the environment May be caused by contaminants (eg smoke or tobacco smoke), excessive physical activity during exercise, stress, or cold air intake.

The term "asthma disease" refers to the characteristics of an individual who, in the case of an individual, suffers from an attack of asthma upon exposure to one or multiple asthma factors. Individuals may be characterized, for example, suffering from allergen-induced asthma, exercise-induced asthma, contaminant-induced asthma, virus-induced asthma or cold-induced asthma.

The terms "chronic obstructive pulmonary disease" and "COPD", used interchangeably herein, reduce the maximum exhalation of the lung and gradually force the lung to be reversible or only minimally reversible to a typical bronchodilator without significant change over several months. Refers to a chronic disorder or combination thereof characterized by emptying. Most commonly, COPD is a combination of chronic bronchitis, i.e. cough and sputum for at least two months and about two years in a row, and airborne, ie alveolar damage. COPD, on the other hand, may include chronic bronchitis with normal air flow, chronic bronchitis with chronic airway disorders (chronic disorder bronchitis), pneumoconiosis, asthma bronchitis and bullous disease, and combinations thereof.

The term "breathing" refers to the process by which oxygen enters the body and the carbon monoxide is released through the body system, including the nose, throat, larynx, trachea, bronchus, and lungs.

The term “respiratory disease or condition” refers to one of several diseases that involve inflammation and particularly affect the components of the bronchial system, including the larynx, bronchus and lung. These diseases include asthma diseases including allergen-induced asthma, exercise-induced asthma, contaminant-induced asthma, cold-induced asthma, stress-induced asthma and virus-induced asthma, chronic bronchitis with normal air flow , Chronic obstructive pulmonary disease, including chronic bronchitis (chronic disorder bronchitis) with airway disorders, pneumonia, bronchitis, and bullous disease, and cystic fibrosis, pigeon breeder's disease, farmer's lung, acute respiratory distress syndrome, pneumonia, aspiration Or inhalation injury, fatty embolism in the lungs, acidosis inflammation of the lungs, acute pulmonary edema, acute altitude sickness, after heart surgery, acute pulmonary hypertension, permanent pulmonary hypertension in newborns, prenatal respiratory syndrome, vitreous membrane disease, acute pulmonary embolism, heparin Other inflammatory lung diseases including protamine reactions, sepsis, asthma persistence and hypoxia.

As used herein, the term “amount effective for respiratory disease” refers to a specific amount of therapeutic compound, or a specific amount of therapeutic compound combination in a combination therapy. Such amounts or combined amounts serve the purpose of preventing, avoiding, alleviating or eliminating a respiratory disease or condition.

In one exemplary embodiment of the selective iNOS inhibitor, the treatment is a compound of formula (I) or a pharmaceutically acceptable salt thereof, a compound of formula (II) or a pharmaceutically acceptable salt thereof, a compound of formula (III) or a pharmaceutical thereof Or a pharmaceutically acceptable salt thereof, a compound of formula (V) or a pharmaceutically acceptable salt thereof, a compound of formula (VI) or a pharmaceutically acceptable salt thereof, Or a pharmaceutically acceptable salt thereof, a compound of formula (VIII) or a pharmaceutically acceptable salt thereof, a compound of formula (IX) or a pharmaceutically acceptable salt thereof, and a compound of formula (X) or a pharmaceutically acceptable salt thereof Promoted through acceptable salts.

Formula I

Where

R ¹ is selected from the group consisting of H, halo, and alkyl which may be optionally substituted with one or more halo,

R ² is selected from the group consisting of H, halo, and alkyl which may be optionally substituted with one or more halo,

At least one of R ¹ and R ² contains halo,

R ⁷ is selected from the group consisting of H and hydroxy,

J is selected from the group consisting of hydroxy, alkoxy and NR ³ R ⁴ ,

Wherein R ³ is selected from the group consisting of H, lower alkyl, lower alkylenyl and lower alkynyl,

R ⁴ is selected from the group consisting of H, and a heterocyclic ring wherein at least one of the rings is carbon and from 1 to about 4 heteroatoms are independently selected from oxygen, nitrogen, and sulfur, wherein the heterocyclic ring is heteroarylamino , N-aryl-N-alkylamino, N-heteroarylamino-N-alkylamino, haloalkylthio, alkanoyloxy, alkoxy, heteroaralkoxy, cycloalkoxy, cycloalkenyloxy, hydroxy, amino, thio, Nitro, lower alkylamino, alkylthio, alkylthioalkyl, arylamino, aralkylamino, arylthio, alkylsulfinyl, alkylsulfonyl, alkylsulfonamido, alkylaminosulfonyl, amidosulfonyl, monoalkyl amidosulfonyl , Dialkyl amidosulfonyl, monoarylamidosulfonyl, arylsulfonamido, diarylamidosulfonyl, monoalkyl monoaryl amidosulfonyl, arylsulfinyl, arylsulfonyl, Teraroarylthio, heteroarylsulfinyl, heteroarylsulfonyl, alkanoyl, alkenoyl, aroyl, heteroaroyl, aralkanoyl, heteroarkanoyl, haloalkanoyl, alkyl, alkenyl, alkynyl, alkylene Dioxy, haloalkylenedioxy, cycloalkyl, cycloalkenyl, lower cycloalkylalkyl, lower cycloalkenylalkyl, halo, haloalkyl, haloalkoxy, hydroxyhaloalkyl, hydroxyaralkyl, hydroxyalkyl, Hydroxyheteroaralkyl, haloalkoxyalkyl, aryl, aralkyl, aryloxy, aralkoxy, aryloxyalkyl, saturated heterocyclyl, partially saturated heterocyclyl, heteroaryl, heteroaryloxy, heteroaryloxyalkyl, arylalkyl , Heteroarylalkyl, arylalkenyl, heteroarylalkenyl, cyanoalkyl, dicyanoalkyl, carboxamidoalkyl, dicarboxamidoalkyl, cyanocarboalkoxy Kil, carboalkoxyalkyl, dicaralkoxyalkyl, cyanocycloalkyl, dicyanocycloalkyl, carboxamidocycloalkyl, dicarboxamidocycloalkyl, carboalkoxycyanocycloalkyl, carboalkoxycycloalkyl, di Carboalkoxycycloalkyl, formylalkyl, acylalkyl, dialkoxyphosphonoalkyl, dialkoxyphosphonoalkyl, phosphonoalkyl, dialkoxyphosphonoalkoxy, dialkoxyphosphonoalkoxy, phosphonoalkoxy, dialkoxyphosphonoalkyl Optionally substituted with amino, dialkoxyphosphonoalkylamino, phosphonoalkylamino, dialkoxyphosphonoalkyl, dialkylalkylphosphonoalkyl, guanidino, amidino or acylamino.

Formula II

Where

X is selected from the group consisting of -S-, -S (O)-and -S (O) ₂ , preferably X is -S-,

R ¹² is C ₁ -C ₆ alkyl, C ₂ -C ₆ alkenyl, C ₂ -C ₆ alkynyl, C ₁ -C ₅ alkoxy-C ₁ alkyl and C ₁ -C ₅ alkylthio-C ₁ alkyl, wherein Each of these groups is optionally substituted with one or more substituents selected from the group consisting of -OH-, alkoxy and halogen), preferably R ¹² consists of -OH, alkoxy and halogen C ₁ -C ₆ alkyl optionally substituted with a substituent selected from the group

As for R ¹³ and R ¹⁸ ,

R ¹⁸ is selected from the group consisting of —OR ²⁴ and —N (R ²⁵ ) (R ²⁶ ), and R ¹³ is —H, —OH, —C (O) —R ²⁷ , —C (O) —OR ²⁸ And -C (O) -SR ²⁹ , or

R ¹⁸ is -N (R ³⁰ )-, R ¹³ is -C (O)-, and R ¹⁸ and R ¹³ together with the atoms to which they are attached form a ring, or

R ¹⁸ is -O-, R ¹³ is -C (R ³¹ ) (R ³² )-, wherein R ¹⁸ and R ¹³ together with the atoms attached thereto form a ring,

When R ¹³ is -C (R ³¹ ) (R ³² )-, R ¹⁴ is -C (O) -OR ³³ , otherwise R ¹⁴ is -H,

R ¹¹ , R ¹⁵ , R ¹⁶ and R ¹⁷ are independently —H, halogen, C ₁ -C ₆ alkyl, C ₂ -C ₆ alkenyl, C ₂ -C ₆ alkynyl and C ₁ -C ₅ alkoxy- Selected from the group consisting of C ₁ alkyl,

R ¹⁹ and R ²⁰ are independently selected from the group consisting of -H, C ₁ -C ₆ alkyl, C ₂ -C ₆ alkenyl, C ₂ -C ₆ alkynyl and C ₁ -C ₅ alkoxy-C ₁ alkyl ,

As for R ²¹ and R ²² ,

R ²¹ is selected from the group consisting of -H, -OH, -C (O) -OR ³⁴ and -C (O) -SR ³⁵ , and R ²² is -H, -OH, -C (O) -OR ³⁶ And -C (O) -SR ³⁷ , or

R ²¹ is -O-, R ²² is -C (O)-, and R ²¹ and R ²² together with the atoms to which they are attached form a ring, or

R ²¹ is -C (O)-, R ²² is -O-, and R ²¹ and R ²² together with the atoms to which they are attached form a ring,

R ²³ is C ₁ alkyl,

R ²⁴ is selected from the group consisting of —H and C ₁ -C ₆ alkyl, and when R ²⁴ is C ₁ -C ₆ alkyl, R ²⁴ is selected from the group consisting of cycloalkyl, heterocyclyl, aryl and heteroaryl Optionally substituted with one or more residues,

As for R ²⁵ and R ²⁶ ,

R ²⁵ is selected from the group consisting of -H, alkyl and alkoxy, R ²⁶ is -H, -OH, alkyl, alkoxy, -C (O) -R ³⁸ , -C (O) -OR ³⁹ and -C ( O) -SR ⁴⁰ , wherein when R ²⁵ and R ²⁶ are independently alkyl or alkoxy, R ²⁵ and R ²⁶ are independently selected from the group consisting of cycloalkyl, heterocyclyl, aryl and heteroaryl Optionally substituted with one or more residues, or

R ²⁵ is -H and R ²⁶ is selected from the group consisting of cycloalkyl, heterocyclyl, aryl and heteroaryl,

R ²⁷ , R ²⁸ , R ²⁹ , R ³⁰ , R ³¹ , R ³² , R ³³ , R ³⁴ , R ³⁵ , R ³⁶ , R ³⁷ , R ³⁸ , R ³⁹ and R ⁴⁰ are independently composed of -H and alkyl Selected from the group wherein the alkyl is optionally substituted with one or more residues selected from the group consisting of cycloalkyl, heterocyclyl, aryl and heteroaryl,

Any R ¹¹ , R ¹² , R ¹³ , R ¹⁴ , R ¹⁵ , R ¹⁶ , R ¹⁷ , R ¹⁸ , R ¹⁹ , R ²⁰ , R ²¹ , R ²² , R ²³ , R ²⁴ , R ²⁵ , R ²⁶ , R ²⁷ , R ²⁸ , R ²⁹ , R ³⁰ , R ³¹ , R ³² , R ³³ , R ³⁴ , R ³⁵ , R ³⁶ , R ³⁷ , R ³⁸ , R ³⁹ and R ⁴⁰ are independently alkyl, alkenyl, alkyne When the residue is selected from the group consisting of one, alkoxy, alkylthio, cycloalkyl, heterocyclyl, aryl and heteroaryl, the residue is optionally substituted with one or more substituents selected from the group consisting of -OH, alkoxy and halogen .

Formula III

Where

R ⁴¹ is H or methyl and R ⁴² is H or methyl.

Formula IV

Formula V

Where

R ⁴³ is hydrogen, halo, C ₁ -C ₅ alkyl, and C ₁ -C ₅ alkyl substituted with alkoxy or one or more halo,

R ⁴⁴ is hydrogen, halo, C ₁ -C ₅ alkyl, and C ₁ -C ₅ alkyl substituted with alkoxy or one or more halo,

R ⁴⁵ is C ₁ -C ₅ alkyl, or C ₁ -C ₅ alkyl substituted with alkoxy or one or more halo.

Formula VI

Where

R ⁴⁶ is C ₁ -C ₅ alkyl, wherein the C ₁ -C ₅ alkyl is optionally substituted with halo or alkoxy and the alkoxy is optionally substituted with one or more halo.

Formula VII

Where

R ⁴⁷ is hydrogen, halo, C ₁ -C ₅ alkyl, and C ₁ -C ₅ alkyl substituted with alkoxy or one or more halo,

R ⁴⁸ is hydrogen, halo, C ₁ -C ₅ alkyl, and C ₁ -C ₅ alkyl substituted with alkoxy or one or more halo,

R ⁴⁹ is C ₁ -C ₅ alkyl, or C ₁ -C ₅ alkyl substituted with alkoxy or one or more halo.

Formula VIII

Where

R ⁵⁰ is C ₁ -C ₅ alkyl, wherein the C ₁ -C ₅ alkyl is optionally substituted with halo or alkoxy and the alkoxy is optionally substituted with one or more halo.

Formula IX

Where

R ⁵⁰ is selected from the group consisting of hydrogen, halo and C ₁ -C ₅ alkyl, wherein said C ₁ -C ₅ alkyl is optionally substituted with halo or alkoxy, said alkoxy is optionally substituted with one or more halo,

R ⁵¹ is selected from the group consisting of hydrogen, halo and C ₁ -C ₅ alkyl, wherein said C ₁ -C ₅ alkyl is optionally substituted with halo or alkoxy, said alkoxy is optionally substituted with one or more halo,

R ⁵² is C ₁ -C ₅ alkyl, wherein the C ₁ -C ₅ alkyl is optionally substituted with halo or alkoxy, the alkoxy is optionally substituted with one or more halo,

R ⁵³ is selected from the group consisting of hydrogen, halo and C ₁ -C ₅ alkyl, wherein said C ₁ -C ₅ alkyl is optionally substituted with halo or alkoxy, said alkoxy is optionally substituted with one or more halo,

R ⁵⁴ is selected from the group consisting of halo and C ₁ -C ₅ alkyl, wherein the C ₁ -C ₅ alkyl is optionally substituted with halo or alkoxy and the alkoxy is optionally substituted with one or more halo.

Formula X

Where

R ⁵⁵ is C ₁ -C ₅ alkyl, wherein the C ₁ -C ₅ alkyl is optionally substituted with halo or alkoxy and the alkoxy is optionally substituted with one or more halo.

In other exemplary compounds, the inducible nitric oxide synthase selective inhibitor is a compound of Formula (XI) or a pharmaceutically acceptable salt thereof. Compound (XI) has already been described in International Publication No. WO 00/26195, published May 11, 2000, which is incorporated herein by reference.

Formula XI

2S-amino-6-[(1-iminoethyl) amino] -N- (1H-tetrazol-5-yl) hexaneamide, hydrate, dihydrochloride

The present invention also contemplates the use of other selective iNOS inhibitors. By way of example, iNOS selective inhibitors also useful in the present invention are described in US Pat. No. 6,355,689 to Beswick et al., Filed November 29, 2000 and issued March 12, 2002, which is represented by Formula XII: Selective iNOS inhibitors are described and claimed.

Formula XII

Where

R ⁷⁹ is selected from C _1-4 alkyl, C _3-4 cycloalkyl, C _1-4 hydroxyalkyl and C _1-4 haloalkyl.

The description of US Pat. No. 6,355,689 states that R ⁷⁹ is preferably C _1-4 alkyl, most preferably methyl. Particular embodiments and compositions disclosed in US Pat. No. 6,355,689 and useful for use in the methods of the present invention include:

S-((R) -2- (1-iminoethylamino) propyl) -L-cysteine,

S-((S) -2- (1-iminoethylamino) propyl) -L-cysteine,

S-((R / S) -2- (1-iminoethylamino) propyl) -L-cysteine,

S-((R) -2- (1-iminoethylamino) propyl) -D-cysteine,

S-((S) -2- (1-iminoethylamino) propyl) -D-cysteine,

S-((R / S) -2- (1-iminoethylamino) propyl) -D-cysteine,

S-((R / S) -2- (1-iminoethylamino) butyl) -L-cysteine,

S-((R / S) -2- (1-iminoethylamino, 2-cyclopropyl) ethyl) -L-cysteine and

S-((R / S) -2- (1-iminoethylamino, 3-hydroxy) propyl) -L-cysteine,

Or pharmaceutically acceptable salts, solvates or physiologically active derivatives thereof.

Such selective iNOS inhibitors are believed to act by competing with arginine as a substrate for iNOS enzymes. Another measure for the inhibition of iNOS is International Patent Application No. PCT / US98 / 03176, published on August 27, 1998, by Arnaiz et al., Publication No. WO 98/37079 (Berlex Laboratories, Inc.). Richmond, CA 94804-0099 and Pharmacopeia, Inc. Princeton, NJ 08540). The Arnaise application describes inhibitors of iNOS monomer dimerization. The iNOS enzyme is a homodimer and each monomer has a reductase domain to incorporate binding sites for flavin cofactors (FAD and FMN) and NADPH. The reductase domain supplies electrons to the oxidase domain of the other monomers, where L-arginine is oxidized at the active site, which incorporates the heme group (Fe) cytochrome P-450 domain. Tetrahydrobiopterin (BH4) is required for homodimerization and regulates the heme redox state during electron transfer. iNOS monomers are inert and dimerization is required for activity.

Accordingly, in another embodiment of the present invention, the selective iNOS inhibitor is a dimerization inhibitor represented by the compound of formula (XIII), (XIV) or (XV) as a single stereoisomer or mixture thereof, or a pharmaceutically acceptable salt thereof. .

Formula XIII

Formula XIV

Formula XV

In the above formulas,

A is -R ⁵⁶ , -OR ⁵⁶ , C (O) N (R ⁵⁶ ) R ⁵⁷ , P (O) [N (R ⁵⁶ ) R ⁵⁷ ] ₂ , -N (R ⁵⁶ ) C (O) R ⁵⁷ , -N (R ⁷⁶ ) C (O) OR ⁵⁶ , -N (R ⁵⁶ ) R ⁷⁶ , -N (R ⁷¹ ) C (O) N (R ⁵⁶ ) R ⁷¹ , -S (O) _t R ⁵⁶ ,- SO ₂ NHC (O) R ⁵⁶ , -NHSO ₂ R ⁷⁷ , -SO ₂ NH (R ⁵⁶ ) H, -C (O) NHSO ₂ R ⁷⁷ and -CH = NOR ⁵⁶ ,

Each X, Y and Z is independently N or C (R ¹⁹ ),

Each U is N or C (R ⁶⁰ ), where U is only N when X is N and Z and Y are CR ⁷⁴ ,

V is N (R ⁵⁹ ), S, O or C (R ⁵⁹ ) H,

Each W is N or CH,

Q is selected from the group consisting of a direct bond, -C (O)-, -O-, -C (= NR ⁵⁶ )-, S (O) _t and -N (R ⁶¹ )-,

m is 0 or an integer from 1 to 4,

n is 0 or an integer of 1 to 3,

q is 0 or 1,

r is 0 or 1, but when Q and V are heteroatoms, m, q and r cannot all be 0,

A is -OR ⁵⁶ , N (R ⁵⁶ ) C (O) R ⁵⁷ , -N (R ⁷¹ ) C (O) OR ⁵⁷ , -N (R ⁵⁶ ) R ⁷⁶ , -N (R ⁷¹ ) C (O) When N (R ⁵⁶ ) R ⁷¹ , -S (O) _t R ⁵⁶ , where t is 0, or -NHSO ₂ R ⁷⁷ , all of n, q and r cannot be 0, and Q is hetero A and A are -OR ⁵⁶ , N (R ⁵⁶ ) C (O) R ⁵⁷ , -N (R ⁷¹ ) C (O) OR ⁵⁷ , -N (R ⁵⁶ ) R ⁷⁶ , N (R ⁷¹ ) C (O ) N (R ⁵⁶ ) R ⁷¹ , -S (O) _t R ⁵⁶ (where t is 0) or -NHSO ₂ R ⁷⁷ , m and n cannot both be 0,

t is 0, 1 or 2,

Is optionally substituted N-heterocyclyl,

Is optionally substituted carbocyclyl or optionally substituted N-heterocyclyl,

Each of R ⁵⁶ and R ⁵⁷ is hydrogen, optionally substituted C ₁ -C ₂₀ alkyl, optionally substituted cycloalkyl, — [C ₀ -C ₈ alkyl] -R ⁶⁴ ,-[C ₂ -C ₈ alkenyl ] -R ⁶⁴ ,-[C ₂ -C ₈ alkynyl] -R ⁶⁴ ,-[C ₂ -C ₈ alkyl] -R ⁶⁵ (optionally substituted with hydroxy),-[C ₁ -C ₈ ]- R ⁶⁶ (optionally substituted with hydroxy), independently selected from the group consisting of optionally substituted heterocyclyl, or

R ⁵⁶ and R ⁵⁷ are N-heterocyclyl optionally substituted with a nitrogen atom attached to them,

R ⁵⁸ is hydrogen, alkyl, cycloalkyl, optionally substituted aryl, haloalkyl,-[C ₁ -C ₈ alkyl] -C (O) N (R ⁵⁶ ) R ⁵⁷ ,-[C ₁ -C ₈ alkyl] -N (R ⁵⁶ ) R ⁵⁷ ,-[C ₁ -C ₈ alkyl] -R ⁶³ ,-[C ₂ -C ₈ alkyl] -R ⁶⁵ ,-[C ₁ -C ₈ alkyl] -R ⁶⁶ , and hetero or a direct bond with R ⁵⁸ - heterocyclyl selected from the group consisting of (halo, alkyl, alkoxy and imidazole optionally substituted with one or more substituents selected from the group consisting of thiazolyl) or, or Q is -N (R ⁵⁸⁾ R ⁵⁸ may further be aminocarbonyl, alkoxycarbonyl, alkylsulfonyl, monoalkylaminocarbonyl, dialkylaminocarbonyl and -C (= NR ⁷³ ) -NH ₂ , or -QR ⁵⁸ Together with -C (O) OH, -C (O) N (R ⁵⁶ ) R ⁵⁷ or Indicates,

R ⁵⁹ is selected from the group consisting of hydrogen, alkyl, aryl, aralkyl and cycloalkyl,

When A is —R ⁵⁶ or —OR ⁵⁶ , R ⁵⁹ may not be hydrogen; when V is CH, R ⁵⁹ may additionally be hydroxy,

R ⁶⁰ is hydrogen, alkyl, aryl, aralkyl, haloalkyl, optionally substituted aralkyl, optionally substituted aryl, -OR ⁷¹ , -S (O) _t -R ⁷¹ , N (R ⁷¹ ) R ⁷⁶ , N (R ⁷¹ ) C (O) N (R ⁵⁶ ) R ⁷¹ , N (R ⁷¹ ) C (O) OR ⁷¹ , N (R ⁷¹ ) C (O) R ⁷¹ ,-[C ₀ -C ₈ alkyl] -C (H) [C (O) R ⁷¹ ] ₂ and-[C ₀ -C ₈ alkyl] -C (O) N (R ⁵⁶ ) R ⁷¹ ,

R ⁶¹ is hydrogen, alkyl, cycloalkyl,-[C ₁ -C ₈ alkyl] -R ⁶³ ,-[C ₂ -C ₈ ] alkyl] -R ⁶⁵ ,-[C ₁ -C ₈ alkyl] -R ⁶⁶ , Acyl, -C (O) R ⁶³ , -C (O)--[C ₁ -C ₈ alkyl] -R ⁶³ , alkoxycarbonyl, optionally substituted aryloxycarbonyl, optionally substituted aralkoxycarbonyl , Alkylsulfonyl, optionally substituted aryl, optionally substituted heterocyclyl, alkoxycarbonylalkyl, carboxyalkyl, optionally substituted arylsulfonyl, aminocarbonyl, monoalkylaminocarbonyl, dialkylaminocarbonyl , Optionally substituted arylaminocarbonyl, aminosulfonyl, monoalkylaminosulfonyl dialkylaminosulfonyl, arylaminosulfonyl, arylsulfonylaminocarbonyl, optionally substituted N-heterocyclyl, -C ( = NH) -N (CN) R ⁵⁶ , -C (O) R ⁷⁸ -N (R ⁵⁶ ) R ⁵⁷ , -C (O) -N (R ⁵⁶ ) R ⁷⁸ -C (O) OR ⁵⁶ Selected from

Each of R ⁶³ and R ⁶⁴ is optionally substituted with one or more substituents selected from the group consisting of haloalkyl, cycloalkyl (optionally substituted with halo, cyano, alkyl or alkoxy), carbocyclyl (halo, alkyl and alkoxy) And heterocyclyl (optionally substituted with alkyl, aralkyl or alkoxy), and

Each R ⁶⁵ is halo, alkoxy, optionally substituted aryloxy, optionally substituted aralkoxy, optionally substituted -S (O) _t -R ⁷⁷ , acylamino, amino, monoalkylamino, dialkylamino, Independently selected from the group consisting of (triphenylmethyl) amino, hydroxy, mercapto, alkylsulfonamido,

Each R ⁶⁶ is independently selected from the group consisting of cyano, di (alkoxy) alkyl, carboxy, alkoxycarbonyl, aminocarbonyl, monoalkylaminocarbonyl and dialkylaminocarbonyl,

Each of R ⁶⁷ , R ⁶⁸ , R ⁶⁹ , R ⁷⁰ , R ⁷² and R ⁷⁵ is independently hydrogen or alkyl,

Each R ⁷¹ is independently hydrogen, alkyl, optionally substituted aryl, optionally substituted aralkyl or cycloalkyl,

R ⁷³ is hydrogen, NO ₂ or toluenesulfonyl,

Each R ⁷⁴ is independently hydrogen, alkyl (optionally substituted with hydroxy), cyclopropyl, halo or haloalkyl,

Each R ⁷⁶ is independently hydrogen, alkyl, cycloalkyl, optionally substituted aryl, optionally substituted aralkyl, —C (O) R ⁷⁷ or —SO ₂ R ⁷⁷ , or

R ⁷⁶ is N-heterocyclyl optionally substituted with R ⁵⁶ and the nitrogen attached thereto; or

R ⁷⁶ is N-heterocyclyl optionally substituted with R ⁷¹ and nitrogen attached thereto,

Each R ⁷⁷ is independently alkyl, cycloalkyl, optionally substituted aryl or optionally substituted aralkyl,

R ⁷⁸ is an amino acid residue.

Another iNOS dimerization inhibitor, 3- (2,4-difluorophenyl) -6- {2- [4- (1H-imidazol-1-ylmethyl) phenoxy] ethoxy} -2-phenylpyridine (PPA250) is described in Otsusuka et al., J. Phamacol Exp Ther Vol. 303, Issue 1, 52-57, October 2002. PPA250 has the following structure.

Thus, in another embodiment of the present invention, compound PPA250 may be used as a selective iNOS inhibitor.

The following synthesis examples are presented for illustrative purposes and are not intended to limit the scope of the invention. If no isomers are found, single isomers will be obtained using appropriate chromatographic methods.

Example A

(2S, 5E) -2-Amino-6-fluoro-7-[(1-aminoethyl) amino] -5-heptenoic acid, dihydrochloride, monohydrate,

EX-A-1) Trimethylsilyl chloride (107.8 g, 1.00 mol) was added dropwise to a low temperature solution of L-glutamic acid (30.00 g, 0.20 mol) in 300 ml of methanol at 0 ° C. The resulting clear colorless solution was stirred at room temperature. After 18 hours, analysis by thin layer chromatography (30% ethyl acetate in hexanes) showed no starting material remaining. The reaction was then cooled to 0 ° C., triethylamine (134 g, 1.33 moles) was added and a white precipitate formed. Di-t-butyldicarbonate (49 g, 0.23 mol) was added and the mixture was allowed to warm to room temperature. After 3 hours, the solvent was removed and 700 ml of diethyl ether was added. The solution was filtered and the filter cake was rinsed with 500 ml additional diethyl ether. The filtrate was concentrated to 60.8 g (> 95%) of tan oil which was used in the next step without further purification.

EX-A-2) 4-dimethylaminopyridine (5.3 g, 0.44 mole) and di-t-view in a solution of crude product (60 g, 0.22 mole) from EX-A-1 in 300 ml of acetonitrile at room temperature Tyldicarbonate (79.2 g, 0.36 mol) was added. The resulting mixture was stirred for 2 days at room temperature where analysis by thin layer chromatography (25% ethyl acetate in hexanes) showed that most of the starting material was consumed. The solvent was removed under reduced pressure to give 85 g of red oil. The crude material was purified by flash column chromatography on silica gel eluting with ethyl acetate in 1:10 hexanes to give 66.4 g (81%) of the desired di-Boc product as a pale yellow solid.

EX-A-3) DIBAL (1.0 M 64 mL in hexane, 63.9 mmol) was added dropwise to a low temperature solution of EX-A-2 (20 g, 53.3 mmol) in 400 mL of anhydrous diethyl ether over 30 minutes at -78 ° C. It was. After an additional 30 minutes at −78 ° C., the solution was quenched with water (12 mL, 666 mmol) and warmed to room temperature. The opaque mixture was diluted with 350 ml of ethyl acetate, dried over MgSO ₄ and filtered through a pad of Celite. The filtrate was concentrated to a yellow oil. 18.9 g of crude material yellow oil was purified by flash column chromatography on silica gel eluting with ethyl acetate in 1: 4 hexanes to give 13.8 g (75%) of the desired aldehyde product as a clear oil.

EX-A-4) n-butyl lithium (1.6 M in hexanes, 10.9 mL, 17.5 mmol) in a low temperature (-78 ° C) solution of triethyl 2-fluorophosphoacetate (4.67 g, 19.3 mmol) in 20 mL THF Was added. The mixture was stirred for 20 minutes at -78 ° C to yield a light yellow solution. Thereafter, a solution of the product from EX-A-3 (6.0 g, 17.5 mmol) in 5 mL of THF was added via syringe and the resulting mixture was stirred at −78 ° C. for 2 hours, at which time thin layer chromatography (hexane 30% ethyl acetate) showed no starting material remaining. The reaction was quenched with saturated aqueous NH ₄ Cl (30 mL) at −78 ° C. The organic layer was collected and the aqueous layer was extracted with diethyl ether (2 x 50 mL). The combined organics were washed with water (100 mL) and brine (100 mL), dried over MgSO ₄ , filtered and concentrated. 8.6 g of the crude material yellow oil was purified by flash column chromatography on silica gel eluting with ethyl acetate in 1: 4 hexanes to give 6.05 g (79%) of the desired fluoro olefin product as a clear oil. ¹ H NMR and ¹⁹ FNMR confirmed the isolation product had an approximate E: Z ratio of 95: 5.

EX-A-5) To a solution of EX-A-4 (805 mg, 1.86 mmol) in 20 mL methanol was added 200 mg of solid NaBH ₄ (844 mg, 22.4 mmol) at room temperature. The reaction was stirred at ambient temperature for 18 hours, where analysis of thin layer chromatography (30% ethyl acetate in hexane) showed that most of the starting material was consumed. The reaction was quenched with 20 mL saturated aqueous NH ₄ Cl and extracted with ethyl acetate (2 × 35 mL). The glass layers were combined, dried over MgSO ₄ , filtered and concentrated. The crude material, 700 mg of a clear oil, was purified by flash column chromatography on silica gel eluting with ethyl acetate in 1: 4 hexanes to give 353 mg (48%) of the desired allylic alcohol product as clear oil, which was ¹⁹ F NMR confirmed the predominantly containing the desired E-isomer.

EX-A-6) EX-A-5 (1.37 g, 3.5 mmol), polymer-supported tripetylphosphine (3 mmol / g, 1.86 g, 5.6 mmol) and 3-methyl-1, in 50 mL THF, To a mixture of 2,4-oxadiazolin-5-one (450 mg, 4.55 mmol) dimethylazodicarboxylate (820 mg, 5.6 mmol) was added dropwise. The reaction was stirred at rt for 1 h at which time analysis by thin layer chromatography (40% ethyl acetate in hexanes) showed no starting material remaining. The mixture was filtered through celite and the filtrate was concentrated. The resulting yellow oil was partitioned between 30 ml of methylene chloride and 30 ml of water. The organic layer was separated, washed with water (1 × 30 mL) and brine (1 × 30 mL), dried over MgSO ₄ , filtered and concentrated. The crude material, 1.8 g of a yellow oil, was purified by flash column chromatography on silica gel eluting with ethyl acetate in 1: 4 hexanes to give 670 mg (40%) of the desired protected E-allylic amidine product as a clear oil. Obtained, which was found to contain only the desired E-isomer by ¹⁹ F NMR.

EX-A-7) The product from EX-A-6 (670 mg, 1.4 mmol) was dissolved in 25 mL methanol and 25 mL 25% acetic acid in water. Zinc dust (830 mg, 12.7 mmol) was added and the mixture was stirred under sonication for 8 hours, with HPLC analysis showing that only 20% of the starting material remained. Zn powder was filtered from the reaction mixture and the filtrate was stored at -20 ° C for 12 hours. The filtrate was allowed to warm to room temperature, additional glacial acetic acid (7 mL) and zinc powder (400 mg, 6.1 mmol) were added and the mixture was sonicated at room temperature for 1 hour, with HPLC analysis showing 96% product. . The mixture was filtered through celite and the filtrate was concentrated. The crude material was 8 minutes using a gradient of 20-95% A (A: 100% acetonitrile with 0.01% trifluoroacetic acid, B: 100% H ₂ O with 0.01% trifluoroacetic acid) Purified by reverse phase HPLC column chromatography on a YMC Combiprep column eluting over. Fractions containing product were combined and concentrated to give 344 mg (45%) of the desired acetamidine product as the trifluoroacetate salt, which was found to contain only the desired E-isomer by ¹⁹ F NMR.

EX-A-8) A sample of the product of EX-A-7 is dissolved in glacial acetic acid. To this stirred solution is added 10 equivalents of 1N HCl in dioxane. After stirring this solution for 10 minutes at room temperature, all solvents are removed in vacuo to produce the illustrated methyl ester dihydrochloride salt.

Example A) A solution of EX-A-7 (344 mg, 1.4 mmol) in 6 mL of 6.0N HCl was refluxed for 1 h. The solvent was removed in vacuo. The resulting solid was further dissolved in water three more times, concentrated and then dissolved five times in succession in 1.0 N HCl to remove all remaining TFA salt. Upon completion, the desired (2S, 5E) -2-amino-6-fluoro-7-[(1-iminoethyl) amino] -5-heptenoic acid, dihydrochloride product 160 mg (37%) was melting point Obtained as a white solid at 51.5-56.3 ° C., ¹⁹ F NMR showed only containing the desired E-isomer.

Example B

(2S-5E / Z) -2-Amino-6-fluoro-7-[(1-iminoethyl) amino] -5-heptenoic acid, dihydrochloride

EX-B-1) Triethylamine (38.35 g, 0.38 mol) in a low temperature (0 ° C.) solution of L-glutamic acid 5-methyl ester (50.00 g, 0.31 mol) in 400 ml of H ₂ O in 1: 1 dioxane. Then di-t-butyldicarbonate (80.00 g, 0.37 mol) was added. The resulting clear colorless solution was stirred to room temperature. After 18 hours, analysis by thin layer chromatography (30% ethyl acetate in hexanes) showed no starting material remaining. The reaction mixture was quenched with 200 mL of 1.0 N aqueous KHSO ₄ . The organic layer was removed and the aqueous layer was extracted with ethyl acetate (3 x 100 mL). The organic layers were combined, dried over MgSO ₄ , filtered and concentrated to give 72.00 g (89%) of the desired product as a pale yellow oil.

EX-B-2) 4-methylmorpholine (28.11 g, 0.28 mole) and isobutylchloroformate (37.95 g,) in a solution of product from EX-B-1 (72.60 g, 0.28 mole) in 300 mL THF 0.28 mole) was added quickly. The clear yellow solution immediately formed a white precipitate. After 4 minutes, the resulting opaque yellow mixture was filtered, the filtrate was cooled to −10 ° C. and a solution of NaBH ₄ (15.77 g, 0.42 mol) in 200 mL of H ₂ O was added dropwise while maintaining the temperature below 0 ° C. . When all of NaBH ₄ was added, the ice bath was removed and the reaction stirred for 1.5 hours at room temperature. The reaction mixture was quenched with 200 mL of H ₂ O. The organic layer was separated and the aqueous layer was extracted with ethyl acetate (3 x 100 mL). The organic layers were combined, washed with brine, dried over MgSO ₄ , filtered and concentrated to give 58 g (85%) of the desired product as a yellow oil.

EX-B-3) To a solution of EX-B-2 (30.95 g, 0.13 mol) in 100 mL of benzene, 2,2-dimethoxy propane (65.00 g, 0.63 mol) followed by p-toluenesulfonic acid (2.40 g , 12.5 mmol) and 5 g of 3 'molecular sieves were added. The resulting mixture was refluxed for 2 hours, analysis by thin layer chromatography (30% ethyl acetate in hexanes) showed the reaction was complete. The mixture was cooled to room temperature, diluted with diethyl ether (150 mL) and Washed with saturated aqueous NaHCO ₃ (100 mL) followed by brine (100 mL). The organic layer was dried over MgSO ₄ , filtered and concentrated. 30.5 g of crude material yellow oil was purified by flash column chromatography on silica gel eluting with ethyl acetate in 1:10 hexanes to give 15.40 g (42%) of the desired product as pale yellow oil.

EX-B-4) DIBAL (6.0 mL of 1.0M solution in toluene) was added dropwise to a low temperature (-78 ° C) solution of product (1.00 g, 3.00 mmol) from EX-B-3 in 10 mL of methylene chloride. After 30 minutes, the reaction was quenched with saturated potassium sodium tartrate (Rochelle salt) and then warmed to room temperature. The mixture was then filtered through a pad of celite, dried over MgSO ₄ , refiltered and concentrated to give a yellow oil. The crude material, 610 mg of a yellow oil, was purified by flash column chromatography on silica gel eluting with ethyl acetate in 1: 4 hexanes to give 550 mg (71%) of the desired product as a clear oil.

EX-B-5) To an ice-cold (0 ° C.) solution of triethyl 2-fluoro-phosphoshacetate (6.70 g, 27.6 mmol) in 100 ml of methylene chloride was added 1,8-diazabicyclo [5.4.0 ] Undec-7-ene (4.70 g, 31.0 mmol) was added. The mixture was stirred for 1 h at 0 ° C. to yield an orange solution. Then an ice-cold (0 ° C.) solution of product (5.71 g, 22.2 mmol) from EX-B-4 in 15 ml of methylene chloride was added via syringe and the resulting mixture was stirred at ambient temperature for 18 hours. Analysis by thin layer chromatography (30% ethyl acetate in hexanes) showed no starting material remaining. The solvent was removed in vacuo and the resulting mixture was layered between 200 ml of ethyl acetate and 100 ml of water. The organic layer was collected and the aqueous layer was extracted with ethyl acetate (2 x 50 mL). The combined organic layers were washed with 1.0 M aqueous KHSO ₄ (100 mL), water (100 mL) and brine (100 mL), dried over MgSO ₄ , filtered and concentrated to afford the desired fluoro olefin product as a yellow oil. (8.0 g). ¹ H NMR and ¹⁹ F NMR showed that the isolated product had an approximate Z: E ratio of 70:30.

This mixture was used crude without further purification.

EX-B-6) LiBH ₄ (2.0M in THF, 12.7 ml, 25.0 mmol) in a ice-cold (0 ° C.) solution of product (8.0 g, 23.0 mmol) from EX-B-5 in 70 ml THF. ) Was added. The reaction mixture was stirred at ambient temperature for 18 hours, whereby analysis by thin layer chromatography (30% ethyl acetate in hexanes) showed no starting material remaining. THF was removed and the resulting mixture was dissolved in methylene chloride. After cooling to 0 ° C., the reaction was quenched by the slow addition of 1.0M aqueous KHSO ₄ . The mixture was then extracted with ethyl acetate (3 x 50 mL). The organic layers were combined, dried over MgSO ₄ , filtered and concentrated. 8.0 g of crude material clear oil was purified by flash column chromatography on silica gel eluting with ethyl acetate in 1: 4 hexanes to afford 900 mg (13%) of the desired product as a clear oil.

EX-B-7) To an ice-cold (0 ° C.) solution of the product from EX-B-6 (950 mg, 3.1 mmol) in 5 mL of pyridine was added methanesulfonyl chloride (390 mg, 3.4 mmol). The reaction was stirred at 0 ° C. for 5 minutes, then warmed to room temperature and stirred for 3 hours, at which time analysis by thin layer chromatography (30% ethyl acetate in hexanes) showed no starting material remaining. The reaction was diluted with diethyl ether (10 mL) and washed with saturated aqueous NaHCO ₃ (20 mL) followed by 1.0 M citric acid (20 mL). The organic layer was dried over MgO ₄ , filtered and concentrated to give 500 mg (51%) of the desired allyl chloride product as a white solid. This product was used next without further purification. LCMS: m / z = 344.1 [M + Na] ⁺

EX-B-8) Potassium phthalimide (290 mg, 1.57 mmol) was added to a stirred solution of the product from EX-B-7 (440 mg, 1.37 mmol) in 10 mL of DMF. The resulting mixture was heated under reflux for 18 hours, whereby analysis by thin layer chromatography (30% ethyl acetate in hexanes) showed no starting material remaining. The cooled mixture was diluted with 30 mL of water, extracted twice with ethyl acetate (30 mL), dried over MgSO ₄ , filtered and concentrated to give 540 mg (91%) of the desired product as a yellow oil.

EX-B-9) The product from EX-B-8 (600 mg, 1.38 mmol) was dissolved in 8 ml of acetic acid and 2 ml of water. The mixture was stirred overnight at room temperature, where analysis by thin layer chromatography (30% ethyl acetate in hexanes) showed no starting material remaining. The solution was concentrated under a stream of nitrogen and the crude product was purified by flash column chromatography on silica gel eluting with ethyl acetate in 1: 2 hexanes to give 248 mg (63%) of the desired product as a white solid.

EX-B-10) Pyridinium dichromate (1.14 g, 3.03 mmol) was added to a stirred solution of the product from EX-B-9 (237 mg, 0.605 mmol) in 6 mL of DMF. The solution turned dark orange and stirred at room temperature for 18 hours, at which time it was poured into 20 mL of H ₂ O. The mixture was extracted with ethyl acetate (4 × 25 mL). The combined organic layers were washed with 5% aqueous KHCO ₃ (3 × 25 mL). The aqueous layer was acidified to pH 3 with 1.0 M KHSO ₄ and then extracted with ethyl acetate (3 × 50 mL). The combined organic layers were concentrated to give 235 mg (95%) of the desired amino acid product. The resulting white solid was used crude without further purification. LCMS: m / z = 429.1 [M + Na] ⁺ .

EX-B-11) To a stirred solution of the product from EX-B-10 (230 mg, 0.56 mmol) in 7 ml of ethanol was added hydrazine hydrate (70 mg, 1.13 mmol) and the resulting solution was refluxed for 2 hours. To form a white precipitate. The solvent was removed in vacuo. The resulting white solid was dissolved in 8 ml of water and acidified to pH 4 with glacial acetic acid. Then cooled in an ice bath and filtered. The filtrate was concentrated to give 136 mg (87%) of the desired allyl amine product as yellow crystals which were used in the next step without further purification. LCMS: m / z = 277.1 [M + H] ⁺ .

EX-B-12) Into a stirred solution of the product from EX-B-11 (136 mg, 0.50 mmol) in 6 ml of DMF was diluted ethyl acetimidate (252 mg, 2.04 mmol) in three portions at 1.5 hour intervals. Added. After the addition was complete, the mixture was stirred at rt overnight. The pink solution was filtered off and the filter cake was washed with water. The solvent was removed in vacuo and the resulting yellow oil was eluted with a 7-minute gradient of 1-50% A (A: 100 acetonitrile containing 0.05% TFA, B: 100 water containing 0.05% TFA) Purification was performed by reversed phase HPLC using prep ODS-A semi-prep column. Fractions containing product were combined and concentrated to give about 50 mg of the desired acetamidine product as trifluoroacetate salt, which was used in the next step. LCMS: m / z = 318.2 [M + H] ⁺ .

Example B) The product from EX-B-2 was dissolved in 6 ml of 6.0N HCl and stirred at room temperature for 1 hour. The solvent was removed in vacuo. The resulting solid was dissolved in water and concentrated three more times to remove the TFA salt. ^{If 19} F NMR indicates complete removal of TFA, the product is dried under vacuum to yield the desired (2S, 5E) -2-amino-6-fluoro-7-[(1-imioethyl) amino] -5- 20:80 E containing heptenoic acid, dihydrochloride and (2S, 5Z) -2-amino-6-fluoro-7-[(1-iminoethyl) amino] -5-heptenoic acid, dihydrochloride 30 mg (20%, combined yield over 2 steps) of a: Z mixture was obtained as a foamable transparent solid.

Example C

(2S, 5Z) -2-Amino-6-fluoro-7-[(1-iminoethyl) amino] -5-heptenoic acid, dihydrochloride

EX-C-1) Triethyl 2-fluoro-phosphonoacetate (3.54 g, 14.6 mmol) was dissolved in 20 ml of CH ₂ Cl ₂ at 0 ° C. and treated with 1,8-diazabicyclo [5.4.0] Dec-7-ene (2.4 mL, 16.4 mmol) was added. The mixture was stirred at 0 ° C. for 20 minutes to yield an orange solution. Then a solution of aldehyde product (4.04 g, 11.7 mmol) from EX-A-3 was added at 0 ° C. and the resulting brown mixture was stirred at rt overnight, at which time LCMS showed no starting material remaining. . Solvent was removed and the residue was partitioned between water (60 mL) and ethyl acetate (120 mL). The organic layer was collected and the aqueous layer was extracted with ethyl acetate (2 x 50 mL). The combined organic layers were washed with water (60 mL) and 10% aqueous KHSO ₄ (60 mL), dried over MgSO ₄ , filtered and concentrated. 5.7 g of the crude material, orange oil, was purified by flash column chromatography on silica gel eluting with 10% ethyl acetate in hexanes to give 3.5 g (69%) of the desired fluoro olefin product as a clear oil. ¹ H NMR and ¹⁹ F NMR showed that the isolated product had a Z / E ratio of 70:30.

EX-C-2) The ester product (3.5 g, 8.1 mmol) from EX-C-1 was dissolved in 80 mL of methanol at room temperature and then solid NaBH ₄ (3 g, 80 mmol) was added in portions. The mixture was stirred at rt for 18 h, with HPLC analysis showing that the reaction was at least 90% complete. The reaction was quenched with saturated NH ₄ Cl. The product was extracted with ethyl acetate and dried over Na ₂ SO ₄ . The organic layer was evaporated to give 3.2 g of crude product as a colorless oil, which was purified by Biotage flash column chromatography, eluting with 20-30% ethyl acetate in hexane to give Z / E of fluoro olefin product as clear oil. 0.41 g (13%) of the desired purity of the Z-isomer product (Z: E = 97: 3 by ¹⁹ F NMR) with 2.11 g (67%) of the mixture was obtained as a clear oil.

EX-C-3) Z-alcohol product (390 mg, 1 mmol) and 3-methyl 1,2,4-oxadiazolin-5-one (130 mg, 1.3 mmol) from EX-C-2 were THF Dissolved in 20 ml. Polymer supported-PPh ₃ was then added to the solution and the mixture was slowly stirred for 10 minutes. Thereafter, diethyl azodicarboxylate was added dropwise and the mixture was stirred at room temperature for 1 hour, at which time LCMS analysis showed that a product had formed and no starting material was present. The polymer was filtered through a pad of celite and the pad was washed with THF. The filtrate was evaporated to afford 1.0 g of crude product, which was purified by biotage flash column chromatography, eluting with 20-30% ethyl acetate in hexane, to give 500 mg of product, which was obtained as a slight didrazide byproduct. Contaminated This material was further purified by biotage column chromatography eluting with 98: 2: 0.01 methylene chloride: methanol: ammonium hydroxide to afford 180 mg (38%) of the desired protected amidine product as a clear oil. ¹⁹ F NMR showed only the desired Z-isomer.

EX-C-4) The product from EX-C-3 (88 mg, 0.19 mmol) was dissolved in 4 mL of 25% acetic acid in water containing a few drops of methanol, followed by Zn powder (109 mg, 1.67 mmol). Added. The mixture was stirred under sonication for 3 hours. Zn was filtered through a pad of celite and the pad was washed with water. The filtrate was evaporated to dryness to afford the crude product, which was eluted for 8 minutes with a gradient of 20 to 80% A (A: 100% ACN with 0.01% TFA, B: 100% H ₂ O with 0.01% TFA). Purified by reverse phase HPLC column chromatography on YMC Combiprep column. The desired product was collected in two fractions and the combined fractions were concentrated. ¹⁹ F NMR gave the product as a colorless oil as a mixture of trifluoroacetate salts containing only the desired Z-isomer, of which 30% was mono Boc-protected product.

Example C) The combined mono- and di-BOC products from EX-C-4 were dissolved in 30 mL of 6N HCl and the solution was refluxed for 4 hours, with LCMS analysis showing the reaction was complete. Excess HCl and water were removed under vacuum. Upon completion, desired (2S, 5Z) -2-amino-6-fluoro- [7-[(1-iminoethyl) amino] -5-heptenoic acid, 9 mg of dihydrochloride product (combined during two steps) Yield 40%) was obtained as a light yellow, very hygroscopic foam, which was found to contain only the desired Z-isomers by ¹⁹ F NMR.

Example D

(2S, 5Z) -2-Amino-6-fluoro-7-[(1-iminoethyl) amino] -5-heptenoic acid, trihydrochloride, dihydrate

EX-D-1) The product from EX-D-2 (3.75 g, 10 mmol) was dissolved in 60 mL methanol and solid NaBH ₄ (4 g, 106 mmol) was added in portions over 10 hours at room temperature. HPLC analysis showed approximately 84% reduction. The reaction mixture was quenched with saturated NH ₄ Cl and then extracted three times with ethyl acetate. The combined organic layers were dried over MgSO ₄ , filtered and evaporated to afford 3.2 g of crude product as a yellow oil.

EX-D-2) Alcohol product (3.2 g, 9.0 mmol ) from EX-D-1 was dissolved in 100 mL THF and cooled in an ice bath. Carbon tetrabromide (4.27 g, 12.9 mmol) was added and the resultant The resulting solution was stirred under nitrogen for 30 minutes at 0 ° C. Polymer-supported PPh ₃ was added and the mixture was slowly stirred at 0 ° C. for 1 hour and then stirred overnight at room temperature. The celite pad was washed with THF, and the filtrate was evaporated to afford the crude product which was purified by Biotage flash column chromatography eluting with ethyl acetate in 1: 3 hexanes to give the desired bromo product 2.0. g (54%, combined yield over 2 steps) was obtained as a colorless oil.

EX-D-3) A solution of NaOEt (21% in EtOH, 41.1 ml, 0.11 mol) in 60 ml ethanol was treated with p-methoxy benzenethiol (14.0 g, 0.1 mol) followed by ethyl chlorofluoroacetate ( 18.3 g, 0.13 mol). The mixture was stirred at rt for 2 h and diluted with 250 mL of hexane in 1: 1 ethyl acetate. The organic layer was washed three times with water and dried over Na ₂ SO ₄ . The dried organic layer was evaporated to give 25 g of the crude product which was used next without further purification.

EX-D-4) A solution of crude product (24 g, 0.1 mol) from EX-D-3 in 200 ml of methylene chloride was cooled to -78 ° C and 3-chloroperbenzoic acid (27 g, 0.12 in 200 ml of methylene chloride) Mole). The reaction mixture was slowly warmed to room temperature and stirred overnight, at which time LCMS analysis showed that the product formed and no starting material remained. The solid was filtered off and the filtrate was washed with saturated NaHCO ₃ and NH ₄ Cl. The organic layer was dried over MgSO ₄ and evaporated to give 30 g of orange oil, which was purified by biotage flash column chromatography, eluting with hexane in 2: 1 ethyl acetate to give 17.5 g (70%) of the desired sulfoxide product as off-white. Obtained as an oil.

EX-D-5) A suspension of NaH (60% in mineral oil, 212 mg, 5.3 mmol) in 6 mL of dry DMF was cooled to 0 ° C. under nitrogen and the sulfoxide product from EX-D-4 in 2 mL of DMF ( 1.25 g, 4.8 mmol) solution. After stirring for 20 minutes at room temperature, the mixture was cooled to 5 ° C. and bromo product (2.17 g, 5.3 mmol) from EX-D-2 was added in one portion. The reaction was stirred at room temperature for 3 hours and then heated at reflux at 95 ° C. for 1 hour, with LCMS analysis showing the formation of the product. The mixture was poured into an ice / aqueous NH ₄ Cl mixture. The product was extracted with hexane in 1: 1 ethyl acetate. The organic layer was dried over Na ₂ S0 ₄ and evaporated to afford 3.17 g of crude yellow oil, which was purified by biotage flash column chromatography eluting with 10% ethyl acetate in hexanes to yield 1.05 g of the desired fluoro olefin ester product. (50%) was obtained as a colorless oil. ¹⁹ F NMR showed that the isolated product contained only the desired 95: 5 Z-isomer.

EX-D-6) The ester product (1.05 g, 2.4 mmol) from EX-D-5 was dissolved in methanol at room temperature and the solid NaBH ₄ was added in portions. The mixture was stirred for 18 hours at room temperature, then 2 ml of water was added and the mixture was stirred for an additional 3 hours, with HPLC analysis showing that the reaction was at least 95% complete. The reaction was quenched with saturated NH ₄ Cl. The product was extracted with ethyl acetate and the organic layer was dried over Na ₂ SO ₄ and evaporated to give 0.95 g of crude product as a colorless oil. ¹⁹ F NMR showed that the isolated crude product contained only the desired Z-isomer.

EX-D-7) Alcohol product from EX-D-6 (0.95 g, 2.4 mmol) and 3-methyl-1,2,4-oxadiazolin-5-one (290 mg, 2.9 mmol) were diluted with THF 60 Dissolved in ml. Polymer-bound triphenyl phosphine was added and the mixture was slowly stirred for 10 minutes. Thereafter, dimethyl azodicarboxylate was added dropwise and the mixture was stirred at room temperature for 1 hour, at which time LCMS analysis showed that a product had formed and no starting material remained. The polymer was filtered through a pad of celite and the pad was washed with THF. The filtrate was evaporated to give a residue, which was partitioned between methylene chloride and water. The organic layer was washed twice with water, dried over MgSO ₄ and evaporated to afford 1.3 g of crude product, which was purified by biotage flash column chromatography, eluting with 20-30% ethyl acetate in hexane, to the desired protected amino. 390 mg (34%, combined yield over two steps) of the dean product was obtained as a colorless oil. ¹⁹ F NMR showed that the isolated product contained only the desired Z-isomer.

EX-D-8) The product from EX-D-7 (390 mg, 0.82 mmol) was dissolved in 20 ml of 25% HOAc in water containing 4 ml of methanol and Zn powder (482 mg, 7.42 mmol) was added in two portions. And added in portions. The mixture was stirred under sonication for 3 hours. Zn was filtered through a pad of celite and the pad was washed with water. The filtrate was evaporated to dryness to afford the crude product which was purified by reverse phase HPLC. Fractions containing the desired product were collected, combined and concentrated. The product was obtained as a colorless oil as a mixture of trifluoroacetate salts, which was found to contain only the desired Z-isomer by ¹⁹ F NMR, 30% of which was mono-Boc protected product.

Example D) Mono and di-BOC products from EX-D-8 were dissolved in 80 mL of 6N HCl and the solution was heated at reflux for 1 hour, with LCMS analysis showing the reaction was complete. Excess HCl and water were removed under vacuum to afford the desired (2S, 5Z) -2-amino-6-fluoro-7-[(1-iminoethyl) amino] -5-heptenoic acid, trihydrochloride, di 150 mg of hydrate product (50% combined over two steps) was obtained as a light yellow, very hygroscopic foam.

Example E

(2R, 5E) -2-amino-6-fluoro-7-[(1-iminoethyl) amino] -5-heptenoic acid, dihydrochloride, monohydrate

EX-E-1) Trimethylsilyl chloride is added dropwise at 0 ° C. to a cold solution of D-glutamic acid in methanol. The resulting clear colorless solution is stirred at room temperature until analysis by thin layer chromatography shows that no starting material remains. The reaction is then cooled to 0 ° C., triethylamine is added and a white precipitate is formed. Di-t-butyldicarbonate is added and the mixture is allowed to warm to room temperature. After 3 hours, the solvent is removed and diethyl ether is added. The solution is filtered and the filter cake is rinsed with additional diethyl ether. The filtrate was concentrated to give the desired mono-Boc diester product, which was used for the next step without further purification.

EX-E-2) To a solution of the crude product from EX-E-1 in acetonitrile add 4-dimethylaminopyridine and di-t-butyldicarbonate at room temperature. The resulting mixture is stirred at room temperature until analysis by thin layer chromatography shows that most of the starting material has been consumed. The solvent is removed in vacuo and the resulting residue is purified by flash column chromatography to give the desired di-Boc protected diester product.

EX-E-3) A solution of DIBAL is added dropwise to a low temperature solution of EX-E-2 in anhydrous diethyl ether at -78 ° C. After 30 minutes at −78 ° C., the solution was quenched with water and warmed to room temperature. The resulting opaque mixture was diluted with ethyl acetate, dried over MgSO ₄ and filtered through a pad of celite. The filtrate was concentrated and the resulting residue was purified by flash column chromatography on silica gel to give the desired aldehyde product.

EX-E-4) n-butyl lithium is added to a low temperature (-78 ° C) solution of triethyl 2-fluorophosphonoacetate in THF. The mixture is stirred at -78 ° C to yield a light yellow solution. Thereafter, a solution of the product from EX-E-3 in THF is added via syringe and the resulting mixture is stirred at −78 ° C. until analysis by thin layer chromatography shows that no starting material remains. The reaction is quenched with saturated aqueous NH ₄ Cl at −78 ° C. The organic layer is collected and the aqueous layer is extracted with diethyl ether. The combined organics are washed with water and brine, dried over MgSO ₄ , filtered and concentrated. The crude material is then purified by flash column chromatography on silica gel to give the desired fluoro olefin product.

EX-E-5) To a solution of EX-E-4 in methanol is added solid NaBH ₄ at room temperature. The reaction is stirred at ambient temperature until analysis by thin layer chromatography shows that most of the starting material is consumed. The reaction is quenched with saturated aqueous NH ₄ Cl and extracted with ethyl acetate. The organic layers are combined, dried over MgSO ₄ , filtered and concentrated. The crude material was purified by flash column chromatography on silica gel to give the desired allylic alcohol product.

EX-E-6) Dimethylazodicarboxylate was added to a mixture of EX-E-5, polymer-supported triphenylphosphine and 3-methyl-1,2,4-oxadiazolin-5-one in THF. Add. The reaction mixture is stirred at room temperature until analysis by thin layer chromatography shows that no starting material remains. The mixture is filtered through celite and the filtrate is concentrated. The resulting yellow oil is partitioned between methylene chloride and water. The organic layer is separated, washed with water and brine, dried over MgSO ₄ , filtered and concentrated. The crude material is purified by flash column chromatography on silica gel to give the desired protected E-allylic amidine product.

EX-E-7) The product from EX-E-6 is dissolved in methanol and acetic acid in water. Zinc powder is added and the mixture is stirred under sonication until HPLC analysis shows that little starting material remains. Zn powder was filtered from the reaction mixture through celite and the filtrate was concentrated. The crude material is purified by reverse phase HPLC column chromatography. Fractions containing product are combined and concentrated to give the desired acetamidine product as trifluoroacetate salt.

Example E) A solution of EX-X-7 in 6.0N HCl is refluxed for 1 hour. The solvent is removed in vacuo. The resulting solid was dissolved in water and repeatedly concentrated from 1.0 N HCl to remove all remaining TFA salts, thereby removing the desired (2R, 5E) -2-amino-6-fluoro-7-[(1-iminoethyl ) Amino] -5-heptenoic acid, dihydrochloride product.

Example F

(2S, 5E) -2-amino-6-fluoro-7-[(1-imioethyl) amino] -5-heptenoic acid, dihydrochloride, monohydrate

EX-F-1) To a solution of EX-A-3 (5.0 g, 11.5 mmol) in THF (45 mL) under nitrogen was added dropwise a solution of red-Al (5.22 mL, 17.4 mmol) in 5.6 mL of THF over 30 minutes. It was. The internal temperature was kept below -10 ° C. After 5 minutes, the reaction was quenched with 33.7 mL 1.3 M Na.K tartrate. Toluene (11 mL) was added to the mixture to improve separation. The organic layer was washed with 33.7 mL 1.3 M Na.K tartrate followed by brine (40 mL). The organic layers were combined, dried over MgSO ₄ , filtered and concentrated. A crude material, 3.8 g (84%) of a light yellow oil was used directly in the next step.

EX-F-2) Triethylamine (18.0 g, 0.179 mol) was added to a solution of the product of EX-F-1 (50.0 g, 0.128 mol) in 500 ml of methylene chloride at -10 ° C. A solution of methanesulfonyl chloride (17.5 g, 0.153 mol) in 50 ml of methylene chloride was added slowly to maintain the temperature at -10 ° C. The reaction was stirred at −10 ° C. for 45 minutes at which time thin layer chromatography (50% ethyl acetate in hexane) and LCMS showed that most of the starting material was consumed. The reaction was quenched with 600 mL of 1.0 M citric acid and extracted with ethyl acetate (2 × 400 mL). The organic layers were combined, dried over MgSO ₄ , filtered and concentrated. 70 g of crude material, yellow oil, was used directly in the next step. LCMS: m / z = 492.2 [M + Na].

EX-F-3) Potassium 3-methyl-1,2,4-oxadiazolin-5-OH in a solution of the product of EX-F-2 (70.0 g, 0.128 mol) in 400 ml of dimethyl formamide at room temperature Nate (28.7 g, 0.192 mol) was added. The reaction was stirred at rt for 2.5 h, where thin layer chromatography (30% ethyl acetate in hexane) and LCMS showed the starting material was consumed. The reaction was diluted with 400 mL of water and extracted with ethyl acetate (5 x 400 mL). The organic layers were combined, washed with 400 mL water, 400 mL brine, dried over MgSO ₄ , filtered and concentrated. 70 g of the crude material, yellow oil, was purified by flash column chromatography on silica gel eluting with ethyl acetate in 1: 4 hexanes to give 38 g (63%) of some yellow oil.

EX-F-4) The combination of several repeated preparations of EX-F-3 was purified by HPLC column chromatography on a Merck silica gel MODCOL column with an isocratic flow rate of 500 mL / min with 60:40 MtBE: heptane. . A second purification of 63 g recovered was chiral HPLC column chromatography on a Chiral Pack-AD column running at 10:90 A: B (A: 100% ethanol, B: 100% heptane) at an isocratic flow rate of 550 ml / min. It was graphy. Fractions containing product were combined and concentrated to give 41 g (68%) of the desired protected L, E-allylic amidine product as clear oil, which was determined by ¹⁹ F NMR and chiral chromatography to L and E-isomers. It was found to contain only.

EX-F-5) The product from EX-F-4 (22.5 g, 0.047 mol) was dissolved in 112 ml of methanol. Vigorous stirring was started and zinc powder (11.5 g, 0.177 mmol) was added followed by 225 ml of 40% acetic acid in water. The stirred reaction was placed under reflux (approx. 60 ° C.) for 2.5 hours, with HPLC analysis showing that most of the starting material was consumed. The reaction was cooled, Zn was filtered from the reaction product through celite and the celite was washed well with additional methanol. The filtrate and methanol washes were combined and concentrated. The resulting oily-white solid was washed with methylene chloride (2 × 500 mL), filtered through a pad of celite and an additional 500 mL wash of methylene chloride was performed. The filtrates were combined and concentrated to give a light yellow oil. 39 g of the crude material, light yellow oil, were purified by plug filtration on 200 ml of silica gel, eluting with 80: 19: 1 methanol: methylene chloride: acetic acid to give 13 g (83%) of the desired product.

Example F) A solution of the product of EX-F-5 (22 g, 0.066 mol) in 750 mL 6.0N HCl was refluxed for 45 minutes. The solvent was removed in vacuo. The resulting solid was dissolved in water and concentrated three more times. YMC ODS-AQ column eluting crude material for 30 minutes by pumping 100% isocratic B over 60 minutes, followed by 10 minutes with a gradient of 0-100% A and 20 minutes with 100% A wash Purified by reverse phase HPLC column chromatography on phase (A: 100% H ₂ O with 100% acetonitrile, B: 0.0025% acetic acid). Fractions containing product were combined and concentrated to yield 3.5 g (68%) of the desired acetamidine product as dihydrochloride salt, which was the desired (2S, 5E) -2-amino-6-fluoro-7. It contained only [[(1-imioethyl) amino] -5-heptenoic acid, dihydrochloride product, obtained as a white solid with a melting point of 51.5-56.3 ° C., which only showed the desired E-isomer according to ¹⁹ F NMR. It was found to contain.

Example G

(2S, 5E) -2-Amino-6-fluoro-7-[(1-hydroxyiminoethyl) amino] -5-heptenoic acid

EX-G-1) Gas HCl was bubbled through a stirred low temperature (0 ° C.) solution of the product of EX-F-3 (14 g, 30.3 mmol) in 100 mL methanol for 5 minutes. The resulting dark yellow solution was stirred for an additional 30 minutes, with HPLC showing that the starting material was consumed completely. The resulting mixture was neutralized to pH 8 with saturated NaHCO ₃ and the product was extracted with EtOAc. The organic layer was dried over MgSO ₄ and concentrated to afford the desired amino ester product as dark yellow oil, which was used crude in the next step.

Example G) A solution of the product of EX-G-1 (8 g, 30 mmol) in 70 mL of 2.5N NaOH was stirred for 10 minutes, with HPLC analysis showing that the starting material was consumed completely. The resulting solution was neutralized to pH 7-8 with 12N HCl (approx. 50 mL) and concentrated. The resulting slurry was washed with methanol, filtered to remove salts and concentrated to a brownish oil. Reverse phase HPLC on a YMC ODS-AQ column eluting the crude material by pumping 100% isocratic B over 60 minutes, followed by 10 minutes with a gradient of 0-100% A and 20 minutes with 100% A wash. Purification by column chromatography (A: 100% acetonitrile, B: 100%). Fractions containing product were combined and concentrated to yield 1.0 g (14%) of the desired product as a white solid. The product was recrystallized from hot water and isopropyl alcohol, collected by filtration to obtain pure (2S, 5E) -2-amino-6-fluoro-7-[(1-hydroxyiminoethyl) amino] -5-hepte No acid was obtained as a white crystalline solid. Melting point: 198.00 to 200.00 ° C.

Chiral analysis> 97.7%: CrownPak CR (+) at 0.8 ml / min isocratic containing 100% A (A: aqueous HClO ₄ , pH = 1.5)

Example H

(2S, 5E) -2-amino-6-fluoro-7-[(1-iminoethyl) amino] -N- (1H-tetrazol-5yl) 5-heptenamide, dihydrochloride

EX-H-1) The product from EX-F-3 (6.1 g, 0.013 mol) is dissolved in 4 ml of methanol. Vigorous stirring was started and 10 mL of 6N HCl was added. The stirred reaction was left under reflux (approximately 60 ° C.) for 18 hours at which time HPLC analysis indicated that most of the starting material was consumed. The reaction was cooled and concentrated to yield 3.3 g (100%) of orange oil. LCMS: m / z = 282 [M + Na] ⁺ .

EX-H-2) The product from EX-F-1 (3.3 g, 0.013 mol) was dissolved in 12 mL of 1: 1 H ₂ O: dioxane. Stirring was started and triethylamine (1.95 g, 0.019 mol) was added. The reaction was cooled to 0 ° C. and di-t-butyldicarbonate (3.4 g, 0.016 mol) was added. The reaction was allowed to warm to room temperature, at which point acetonitrile (4 mL) was added to dissolve the solids. The reaction was stirred for 18 hours at room temperature, where HPLC analysis indicated that most of the starting material was consumed. The reaction was quenched with 1.0N KHSO ₄ (25 mL), extracted with ethyl acetate (3 × 50 mL), and the organic layer was dried over MgSO ₄ and concentrated. 3.5 g of the crude material, dark oil, was purified by flash chromatography, eluting with 4: 95: 1 methanol: methylene chloride: acetic acid to give 2.4 g (52%) of the desired product as a light yellow oil. LCMS: m / z = 382 [M + Na] ⁺ .

EX-H-3) The product from Ex-H-2 (2.4 g, 0.007 mol) was dissolved in 13 mL THF. Stirring was started and 5-aminotetrazole monohydrate (0.83 g, 0.008 mol) was added, followed by 1,3-diisopropylcarbodiimide (1.0 g, 0.008 mol). The resulting mixture was stirred at room temperature for 3 hours, with HPLC showing that most of the starting material was consumed. 12 ml of water was added to the reaction and THF was removed by vacuum distillation. Ethanol (30 mL) was added and the reaction heated under reflux. After 15 minutes under reflux, the reaction was cooled to -10 ° C, at which time the desired product precipitated out of solution. The product was collected by filtration to give 1.25 g (50%) of a white solid. LCMS: m / z = 449 [M + Na] ⁺ .

EX-H-4) The product of EX-H-3 (1.0 g, 0.0023 mol) was dissolved in 5 ml of methanol. Vigorous stirring was started and 10 ml of 40% acetic acid in water followed by zinc powder (0.5 g, 0.008 mol). The stirred reaction was left under reflux (approximately 60 ° C.) for 1.5 hours, with HPLC analysis showing that most of the starting material was consumed. The reaction was cooled and Zn was filtered through celite from the reaction mixture and the celite was washed with additional methanol. The filtrate and methanol washes were combined and concentrated. YMC ODS-AQ column, eluting the resulting oily-white solid with 100% isocratic B by pumping over 60 minutes, followed by 10 minutes with a gradient of 0-100% A and 20 minutes with 100% A wash. Purified by reverse phase HPLC column chromatography on phase (A: 100% acetonitrile, B: 100% H ₂ O with 0.0025% acetic acid). Fractions containing product were combined and concentrated to yield 0.390 g (44%) of the desired acetamidine product as a white solid. LCMS: m / z = 407.3 [M + Na].

Example H) The product from EX-H-4 (0.30 g, 0.780 moles) was dissolved in 5 mL concentrated HOAc. To this was added 1 ml of 4N HCl in dioxane. The reaction was stirred for 5 minutes at room temperature. The solvent was removed in vacuo. The resulting solid was dissolved in water and concentrated three more times. HPLC showed large amounts of starting material. The solid was dissolved in 1N HCl and stirred for 3 hours, with HPLC showing that most of the starting material was consumed. The solution was concentrated to give 290 mg (98%) of the desired acetamidine product as dihydrochloride salt. LCMS: m / z = 285.1 [M + H].

Example I

S- [2-[(1-iminoethyl) amino] ethyl] -2-methyl-L-cysteine, dihydrochloride

Example I-1) (2R, 4R) -Methyl-2-t-butyl-1,3-thiazoline-3-formyl-4-carboxylate

Jeanguenat and Seebach, J. Chem. Soc. Perkin Trans . 1, 2291 (1991), and Pattenden et al. , Tetrahedron , 49, 2131 (1993). (R) -Cysteine methyl ester hydrochloride (8.58 g, 50 mmol) fivalaldehyde (8.61 g, 100 mmol) and triethylamine (5.57 g, 55 mmol) were refluxed in pentane (800 mL) to provide The water was removed continuously using a stark trap. The mixture was filtered and evaporated. The resulting niazolidine (9.15 g, 45 mmol) and sodium formmate (3.37 g, 49.5 mmol) were stirred in formic acid (68 mL), treated with acetic anhydride (13 mL, 138 mmol) and 0-5 ° C. Was added dropwise over 1 hour. The solution was allowed to warm to room temperature and stirred overnight. The solvent was evaporated and the residue neutralized with aqueous 5% NaHCO ₃ and extracted with ether (× 3). The combined organic layers were dried (anhydrous MgSO ₄ ), filtered and evaporated to afford the title compound (8.65 g) which crystallized from hexane-ether as white crystals (80% total, 8: 1 mixture of isoforms).

Example-I-2) (2R, 4R) -Methyl-2-t-butyl-1,3-thiazoline-3-formyl-4-methyl-4-carboxylate

To a solution of the product of Example-I-2 in (2R, 4R) -methyl-2-t-butyl-1,3-thiazoline-3- in anhydrous tetrahydrofuran (130 mL) at -78 ° C. under N _2. Formyl-4-carboxylate (8.65 g, 37.4 mmol) was added and the mixture was stirred for 5 minutes. Lithium bis (trimethylsilyl) amide in 1M tetrahydrofuran (37.5 mL) was added and the mixture was stirred for 30 minutes. Methyl iodide (5.84 g, 41.1 mmol) was added and the mixture was kept at -78 ° C for 4 hours, then warmed to room temperature under constant stirring. The solvent was evaporated in vacuo and brine and ethyl acetate were added. The aqueous phase was extracted three times with EtOAc and the combined organic layers were washed with 10% KHSO ₄ , water and brine. They were then dried (anhydrous MgSO ₄ ), filtered and all solvents stripped under reduced pressure. The remaining oil was chromatographed on silica with 1-10% EtOAC / hexanes to give the title compound (5.78 g, 63%, 2.4: 1 isomeric mixture).

Retention time on Daicel Chemical Industries Chiracel OAS column 16.5 minutes, 10-40% IPA / hexanes 0-25 minutes,> 95% ee.

Example-I-3) (2R) 2-Methyl-L-cysteine hydrochloride

Product of Example-I-2, of (2R, 4R) -methyl-2-t-butyl-1,3-thiazoline-3-formyl-4-methyl-4-carboxylate (5.7 g, 23.2 mmol) The product was stirred with 6N HCl (100 mL) under N ₂ and maintained for 2 days under vigorous reflux. The solution was cooled, washed with EtOAc and evaporated to afford the product (2R) 2-methyl-cysteine hydrochloride (3.79 g, 95%) as a light yellow powder.

Example-I-4) S- [2-[[(1,1-dimethylethoxy) carbonyl] amino] ethyl] -2-methyl-L-cysteine trifluoroacetate

Sodium hydride (2.6 g, 60% in mineral oil, 65 mmol) was added to an oven dried vacuum-cooled RB flask containing oxygen-free 1-methyl-2-pyrrolidinone (5 mL). The mixture was cooled to -10 ° C and stirred under N ₂ . The product of Example-I-3, 2-methyl-L-cysteine hydrochloride (3.6 g, 21.0 mmol), was added in portions of oxygen-free 1-methyl-2-pyrrolidinone (25 mL). After all H ₂ generation has ceased, 2-[(1,1-dimethylethoxycarbonyl) -amino] elibromide (4.94 g, 21 in oxygen-free 1-methyl-2-pyrrolidinone (15 mL) Mmol) was added at -10 ° C. The reaction was then stirred for 4 hours and allowed to warm to room temperature. The solution was neutralized with 1N HCl and 1-methyl-2-pyrrolidinone was removed by evaporation in vacuo. Appropriate fractions by reverse phase chromatography with 1-20% acetonitrile in 0.05% aqueous trifluoro acetic acid solution were recovered by freeze drying to afford the title compound (5.9 g).

Example-I-5) S- (2-Aminoethyl) -2-methyl-L-cysteine hydrochloride

Product of Example-I-4, S- [2-[[(1,1-dimethylethoxy) carbonyl] amino] ethyl] -2-methyl-L-cysteine trifluoroacetate (5.5 g, 14.0 Mmol) was dissolved in 1N HCl (100 mL) and stirred overnight at room temperature under nitrogen. The solution was removed by lyophilization to give the title S- (2-aminoethyl) -2-methyl-L-cysteine hydrochloride.

Example I) The product of Example-I-5 was dissolved in H ₂ O, the pH was adjusted to 10 with 1N NaOH, and ethyl acetimimidate hydrochloride (1.73 g, 14.0 mmol) was added. The reaction was stirred for 15-30 minutes, the pH was raised to 10 and the process repeated three times. The pH was adjusted to 3 with HCl and the solution loaded onto a washed DOWEX 50WX4-200 column. The column was washed with H ₂ O and 0.25M NH ₄ OH followed by 0.5M NH ₄ OH. Fractions from 0.5 M NH ₄ OH were immediately frozen, combined and lyophilized to give an oil which was dissolved in 1N HCl and evaporated to give the title compound as a white solid (2.7 g).

Example J

2 [[[2-[(1-iminoethyl) amino] ethyl] thio] methyl] -O-methyl-D-serine, dihydrochloride

The procedure and method used in this example was the same as that of Example I, except that methoxymethyl iodide instead of methyl iodide was used in Example-I-2. This procedure gave the title compound as a white solid (2.7 g).

Example K

S-[(1R) -2-[(1-Iminoethyl) amino] -1-methylethyl] -2-methyl-L-cysteine, dihydrochloride

Example-K-1) (S) -1-[(benzyloxycarbonyl) amino] -2-propanol

A solution of (S) -1-amino-2-propanol (9.76 g, 130 mmol) in anhydrous benzene (60 mL) was slowly divided into benzyl chloroformate (10.23 g, 60 mmol) in anhydrous benzene (120 mL) 20 It was added with vigorous stirring under nitrogen atmosphere for minutes. The mixture was stirred at 0 ° C. for 1 h, then warmed to rt and stirred for a further 2 h. After the mixture was washed with water (2 ×) and brine (2 ×), the organic layer was dried over anhydrous MgSO ₄ . All solvents were evaporated to afford the title compound as an oil.

Example-K-2) (S) -1-[(benzyloxycarbonyl) amino] -2-propanol tosylate

The product of Example-K-1 in methylene chloride (60 mL) at 0 ° C., (S) -1-[(benzyloxycarbonyl) amino] -2-propanol (9.74 g, 46.7 mmol) and triethylamine ( To a solution of 7.27 g, 72 mmol) toluene sulfonyl chloride (9.15 g, 48 mmol) in methylene chloride (18 mL) was added slowly while stirring vigorously under nitrogen for 20 minutes. The mixture was allowed to warm to rt and stirred for an additional 36 h under nitrogen. The organic layer was washed with 1N HCl, water, 5% NaHCO ₃ solution, water and brine and then dried over anhydrous MgSO ₄ . Evaporation of all solvents gave a white solid, which was passed through a silica plug with ethyl acetate / hexanes (1: 4) to remove excess toluene sulfonyl chloride and passed through ethylene / hexanes (1: 3) to white crystals. To give the title compound. This material was recrystallized from ethyl acetate / hexanes to give a white needle shape (10.8 g).

The product was used for 5 minutes using a mobile phase of isopropanol / hexane and a gradient of 10% isopropanol followed by 10-40% isopropanol over 25 minutes, all using Regis Technologies Inc. with UV and laser polarimeter detectors. Irradiated on a Rated Perckle Cobalt (R, R) -GEM1 HPLC (Regis Technologies Inc. Perkle Covalent (R, R) -GEM1 HPLC) column. Main peak residence time: 22.2 minutes,> 98% ee.

Example-K-3) S-[(1R) -2- (Benzyloxycarbonylamino) -1-methylethyl] -2-methyl-L-cysteine trifluoroacetate

The product from Example I-3, 2-methyl-L-cysteine hydrochloride (1 g, 6.5 mmol) was added to an oven-dried, N ₂ filled RB flask and oxygen-free 1-methyl-2-pyrroli It was dissolved in deanone (5 mL) and the system was cooled to 0 ° C. Sodium hydride (0.86 g, 60% in mineral oil, 20.1 mmol) was added and the mixture was stirred at 0 ° C. for 15 minutes. The product of Example-K-2, dissolved in oxygen-free 1-methyl-2-pyrrolidinone (10 mL), (2S) -1-[(N-benzyloxycarbonyl) amino] -2-propanol tosyl A solution of rate (2.5 g, 7 mmol) was added over 10 minutes. After 15 minutes at 0 ° C., the reaction mixture was stirred for 4.5 hours at room temperature. The solution was then acidified to pH 4 with 1N HCl and 1-methyl-2-pyrrolidinone was removed in vacuo. Reverse phase chromatography using 20-40% acetonitrile in 0.05% aqueous trifluoro acetic acid solution gave 0.57 g of the title compound, which was recovered by freeze drying.

Example-K-4) S-[(1R) -2-Amino-1-methylethyl] -methyl-L-cysteine hydrochloride

Product of Example-K-3, S-[(1R) -2- (benzyloxycarbonylamino) -1-methylethyl] -2-methyl-L-cysteine trifluoroacetate (0.5 g, 1.14 mmol) Was dissolved in 6N HCl and refluxed for 1.5 h. The mixture was then cooled to rt and extracted with EtOAc. The aqueous layer was concentrated in vacuo to afford the title compound, (2R, 5R) -S- (1-amino-2-propyl) -2-methyl-cysteine hydrochloride (0.29 g), which was used without further purification.

Example K) The product of Example-K-4, S-[(1R) -2-amino-1-methylethyl] -2-methyl-L-cysteine hydrochloride (0.2 g, 0.76 mmol), was dissolved in H ₂ O. Dissolve in 2 ml, adjust the pH to 10.0 with 1N NaOH, add ethyl acetimidate hydrochloride (0.38 g, 3 mmol) in four portions over 10 minutes, and adjust pH to 10.0 with 1N NaOH as needed. Adjusted. After 1 hour, the pH was adjusted to 3 with 1N HCl. The solution was loaded on a washed DOWEX 50WX4-200 column. The column was washed with H ₂ O and 0.5N NH ₄ OH. The basic fractions were pooled and evaporated to dryness in vacuo. The residue was acidified with 1N HCl and concentrated to Example K title compound (49 mg).

Example L

S-[(1S) -2-[(1-Iminoethyl) amino] -1-methylethyl] -2-methyl-L-cysteine, dihydrochloride

The procedures and methods used herein are the same as in Example K, except that (R) -1-amino-2-propanol was used instead of (S) -1-amino-2-propanol in Example-K-1. This gave the title compound, S-[(1S) -2-[(1-iminoethyl) amino] -1-methylethyl] -2-methyl-L-cysteine hydrochloride.

Example M

S- [2- (1-Iminoethyl) amino) ethyl] -2-ethyl-L-cysteine, dihydrochloride

The procedure and method used for this synthesis was the same as that used in Example I, except that ethyl triflate was used instead of methyl iodide in Example-I-2. The title compound was purified (20% yield) using reverse phase chromatography using a gradient of 10-40% acetonitrile in water.

Example N

2-[[[[2- (1-imioethyl) amino] ethyl] thio] methyl] -D-valine, dihydrochloride

Example-N-1) Isopropyl Triplate

Aged silver triplate (25.25 g, 98.3 mmol) in diethyl ether (300 mL) under nitrogen was treated with isopropyl iodide (16.54 g, 98.5 mmol) in ether (200 mL) over 15 minutes. The mixture was stirred for 10 minutes and then filtered. The filtrate was distilled off under reduced pressure. The distillate was re-distilled at ambient pressure to remove most of the diethyl ether, leaving a mixture of the title isopropyl triplate-diethyl ether (84:16 (weight)) (15.64 g, 70% modified) as a colorless liquid. . ¹ H NMR (CDCl ₃ , 400 MHz) δ 1.52 (d, 6H), 5.21 (seven lines, 1H).

The procedure and method used here was the same as that used in Example I, except that the isopropyl triflate replaced methyl iodide in Example-I-2. The crude title product was purified by reverse phase chromatography using a gradient eluent of 10-40% acetonitrile in water.

Example O

S- [2- (1-Imioethylamino) ethyl] -2-methyl- (D / L) -cysteine, bistrifluoroacetate

Example-O-1) S- (2-aminoethyl) -L-cysteine, methyl ester

A 10 g (50 mmol) sample of S- (2-aminoethyl) -L-cysteine was dissolved in 400 ml of methanol. This cooled solution was bubbled with anhydrous HCl for 30 minutes. After stirring at room temperature overnight, the solution was concentrated to give 12.7 g of the title compound.

Example-O-2) N- {4-Chlorophenyl) methylene] -S- [2-[[(4-chlorophenyl) methylene] amino] ethyl] -L-cysteine, methyl ester

The product of Example-O-1, a sample of 12.7 g (50 mmol) of S- (2-aminoethyl) -L-cysteine methyl ester, was dissolved in acetonitrile. To this solution was added 12.2 g (100 mmol) of anhydrous MgSO _{4, 14} g (100 mmol) of 4-chlorobenzaldehyde and 100 mmol of triethylamine. The mixture was stirred for 12 hours, concentrated to a small volume and diluted with 500 ml of ethyl acetate. The organic solution was washed successively with (0.1%) NaHCO ₃ , (2N) NaOH and brine solution. The organics were dried (anhydrous MgSO ₄ ), filtered and concentrated to afford 7.5 g of the title compound. [M + H ⁺ ] = 179.

Example-O-3) N- [4-Chlorophenyl) methylene] -S- [2-[[(4-chlorophenyl) methylene] amino] ethyl] -2-methyl-D / L-cysteine methyl ester

Product of Example-O-2 in dry THF, N- {4-chlorophenyl) methylene] -S- [2-[[(4-chlorophenyl) methylene] amino] ethyl] -L-cysteine methyl ester (7.5 g, 17 mmol) was treated with 17 mmol of sodium bis (trimethylsilyl) amide at −78 ° C. under nitrogen followed by 2.4 g (17 mmol) of methyl iodide. The solution was kept at −78 ° C. for 4 hours and then warmed to room temperature under continuous stirring. The solvent was evaporated in vacuo and brine and ethyl acetate were added. The aqueous phase was extracted with 3xEtOAc and the combined organic layers washed with 10% KHSO ₄ , water and brine, then dried (anhydrous MgSO ₄ ), filtered and evaporated to afford the title compound.

Example-O-4) S- (2-Aminoethyl) -2-methyl-D / L-cysteine, hydrochloride

Product of Example-O-3, N- [4-chlorophenyl) methylene] -S- [2-[[(4-chlorophenyl) methylene] amino] ethyl] -2-methyl-D / L-cysteine methyl A sample of ester (4.37 g, 10 mmol) was stirred overnight with 2N HCl, heated and the solution was washed with ethyl acetate (× 3). The aqueous solution was lyophilized to afford the title compound.

Example O) The product of Example-O-4, S- (2-aminoethyl) -2-methyl-D / L-cysteine dihydrochloride (2.5 g (10 mmol) of sample) was dissolved in H ₂ O , pH was adjusted to 1 N NaOH to 10. Ethyl acetamidate hydrochloride (1.24 g, 10.0 mmol) was added to the reaction mixture The reaction was stirred for 15-30 minutes, pH was raised to 10, This procedure was repeated three times: pH was lowered to HCl and the solution was evaporated The residue was purified on reverse phase HPLC with H ₂ O containing 0.05% trifluoroacetic acid as mobile phase to give Example O title compound. M + H = 220.

Example P

(2R) -2-amino-3 [[2-[(1-iminoethyl) amino] ethyl] sulfinyl] -2-methylpropanoic acid, dihydrochloride

A solution of S- [2-[(1-iminoethyl) amino) ethyl] -2-methyl-L-cysteine, dihydrochloride (Example I, 0.2 g, 0.73 mmol) in 3 ml of water was stirred, Cool to 0 ° C. and add 0.3 ml of a solution of 3% H ₂ O ₂ (0.8 ml, 0.73 mmol) in formic acid (0.4 ml, 0.73 mmol). The low temperature bath was removed and the reaction mixture was stirred for 48 hours at room temperature. The solution was concentrated in vacuo, diluted with water (10 mL) and concentrated again to give crude sulfone. This residue was chromatographed (C-18 reverse phase, using H ₂ O containing 0.05% trifluoroacetic acid as mobile phase) to give pure sulfone. The sulfone was treated with 1M HCl (10 mL) and concentrated in vacuo to give 140 mg of a mixture of the two title compound diastereomers as colorless oil of HCl salt.

Example Q

(2R) -2-amino-3 [[2-[(1-iminoethyl) amino] ethyl] sulfonyl] -2-methylpropanoic acid dihydrochloride

A solution of S- [2-[(1-iminoethyl) amino] ethyl] -2-methyl-L-cysteine dihydrochloride, the product of Example I (0.15 g, 0.54 mmol) in 2 ml of water was brought to 0 ° C. Was cooled down and a solution of 3% H ₂ O ₂ (1.6 mL, 1.46 mmol) in formic acid (0.8 mL, 14.6 mmol) was added. The low temperature bath was removed and the reaction mixture was stirred for 18 hours at room temperature. The solution was concentrated in vacuo, diluted with 10 mL of water and concentrated again to give crude sulfoxide. The residue was diluted with 4 mL of water and adjusted to pH 9 with 2.5N NaOH. Acetone (5 mL) was added followed by Boc ₂ O (0.2 g) and the reaction was stirred at rt for 48 h. The reaction mixture was adjusted to pH 6 with 1M HCl and concentrated in vacuo. This residue was chromatographed (C-18 reverse phase; 40-50% ACN: H ₂ O, 0.05% TFA) to give pure Boc protected material. Fractions were concentrated in vacuo and the residue was treated with 1NHCl (3 mL) for 1 h. The solution was concentrated to give 30 mg of the title compound as a colorless oil.

Example R

(2S, 5Z) -2-Amino-6-methyl-7-[(1-iminoethyl) amino] -5-heptenoic acid, dihydrochloride

Example R-1)

A solution of triethyl-2-phosphonopropionate (6.5 mg, 27.1 mmol) in toluene (60 mL) was treated with 0.5 M potassium bis (trimethylsilyl) amide (50.0 mL, in toluene) and the resulting anion Was condensed with the aldehyde product of Example U-3 by the method of Example U-4 (see Example U below). This gave 8 g of a 3: 7 mixture of the desired Z and E diesters, respectively, after chromatography.

Example R-2)

The product mixture of Example R-1 (850 mg, 2.0 mmol) in Et ₂ O (30 mL) was reduced by diisobutyl aluminum / hydride (DIBAL) for 20 minutes by the method of Example U-5. One crude mixture of the desired E-alcohol and unreduced Z-ester was produced. The mixture was chromatographed on silica gel, eluting with n-hexane: EtOAc (9: 1) to n-hexane: EtOAc (1: 1) to obtain a sample of Z-ester (530 mg) and E-alcohol desired material. Provided.

Example R-3)

The product Z-ester (510 mg, 1.2 mmol) of Example R-2 in Et ₂ O (30 mL) was reduced to diisobutyl aluminum / hydride (DIBAL) for 2 hours by the method of Example U-5. To produce the desired crude Z-alcohol exemplified. This material was chromatographed on silica gel eluting with n-hexane: EtOAc (9: 1) to n-hexane: EtOAc (8: 2) to afford 340 mg of the desired Z-alcohol product.

Example R-4)

Example R-3 (340 mg, 0.9 mmol) CH ₂ Cl ₂ solution (5 mL) of product alcohol was treated with triethylamine (151 mg, 1.5 mmol). To this solution cooled in an ice bath was added a CH ₂ Cl ₂ solution of methanesulfonyl chloride (1.5 mL). After 15 minutes, the ice bath was removed and the reaction stirred for 20 hours at ambient temperature. The reaction mixture was washed with 10% KHSO ₄ , dried over Na ₂ SO ₄ and stripped of all solvents under reduced pressure to yield 350 mg of the desired 8-allylic chloride.

Example R-5)

The suspension of potassium 3-methyl-1,2,4-oxa-dioxazolin-5-one in DMF was reacted with the DMF solution of the product of Example R-4 by the method of Example S-2 below to obtain the material. Manufacture.

Example R-6)

The product of Example R-5 was reacted with zinc in HOAc by the method of Example U-7 to afford amidine.

Example R-7)

The product of Example R-6 was reacted with 4NHCl in dioxane in glacial acetic acid to give amidine.

Example R)

The product of Example R-7 was deprotected to afford amino acid, dihydrochloride.

Example S

(2S, 5E) -2-Amino-6-methyl-7-[(1-iminoethyl) amino] -5-heptenoic acid, dihydrochloride

Example S-1)

The E-alcohol product of Example R-2 (1.3 g, 3.3 mmol) with triethylamine (525 mg, 5.2 mmol) and methanesulfonyl chloride (560 mg, 5.2 mmol) by the method of Example R-4 Reaction gave 1.4 g of the desired E-allylic chloride.

Example S-2)

A suspension of potassium 3-methyl-1,2,4-oxa-diazolin-5-one (460 mg, 3.35 mmol) in 5 ml of DMF was treated with a DMF (15 ml) solution of the product of Example S-1. . The reaction mixture was stirred at 50 ° C. for 17 h and then additionally 50 mg (0.04 mmol) of diazolin-5-one salt. The stirred reaction was continued to heat for an additional 3 hours, then cooled to room temperature and diluted with 180 mL of water. The mixture was extracted with EtOAc and the extract was diluted with 120 mL of n-hexane, washed with water, dried over Na ₂ SO ₄ and all solvents were removed under reduced pressure to give 1.3 g of material.

Example S-3)

The product of Example S-2 (460 mg, 1.0 mmol) was reacted with zinc in HOAc by the method of Example U-7 (see Example U below) to give 312 mg of the desired amidine after HPLC purification. .

Example S)

The product of Example S-3 (77 mg, 0.2 mmol) was deprotected with 2N HCl by the method of Example U to give 63 mg of E-amino acid, dihydrochloride.

Example T

(2S, 5Z) -2-Amino-7-[(1-iminoethyl) amino] -5-heptenoic acid, dihydrochloride

Example T-1) Methyl bis (trifluoroethyl) phosphonoacetate (4.77 g, 15 mmol) and 18-crown-6 23.7 g (90 mmol) were dissolved in 80 mL of dry THF and cooled to -78 ° C. . After adding 30 ml (15 mmol) of potassium bis (trimethylsilyl) amide to this solution, 5.1 g of N, N-diBoc glutamic aldehyde methyl ester from Example U-3 (see Example U below) (14.7 mmol) was added. After stirring at −78 ° C. for 30 minutes, the reaction was quenched with aqueous KHSO ₄ . The reaction mixture was extracted with EtOAc and concentrated to give 2.95 g (49%) of the desired compound. Mass spectrum M + H = 402.

Example T-2) The product from Example T-1 was reduced by the method of Example U-5 to afford the desired compound.

Example T-3) The product from Example T-2 was reacted with 3-methyl-1,2,4-oxadiazolin-5-one by the method of Example U-6 to afford the desired compound. .

Example T-4) The product from Example T-3 was deprotected by the method of Example U-7 to afford the desired compound.

Example T) The product from Example T-4 was dissolved in 2N HCl and heated to reflux. The reaction mixture was cooled and concentrated to yield 0.12 g of the desired product.

Example U

(2S, 5E) -2-amino-7-[(1-iminoethyl) amino] -5-heptenoic acid, dihydrochloride

Example U-1) L-glutamic acid (6.0 g, 40.78 mmol) was dissolved in methanol (100 mL). Trimethylsilyl chloride (22.9 mL, 180 mmol) was added to the reaction mixture at 0 ° C. under nitrogen and stirred overnight. To the reaction mixture was added triethylamine (37 mL, 256 mmol) and di-t-butyldicarbonate (9.8 g, 44.9 mmol) at 0 ° C. under nitrogen and stirred for 2 hours. The solvent was removed and the residue was triturated with ether (200 mL). The ground mixture was filtered. The filtrate was evaporated to oil and chromatographed on silica, eluting with ethyl acetate and hexanes to give mono boc L-glutamic diester (10.99 g, 98%).

Example U-2) Mono boc L-glutamic acid (10.95 g, 39.8 mmol) was dissolved in acetonitrile (130 mL). 4-dimethylaminopyridine (450 mg, 3.68 mmol) and di-t-butyldicarbonate (14.45 g, 66.2 mmol) were added to the reaction mixture and stirred for 20 hours. The solvent was evaporated and the residue was chromatographed on silica, eluting with ethyl acetate and hexanes to give di-boc-L-glutamic diester (14.63 g, 98%).

Example U-3) The product from Example U-2 (10.79 g, 28.7 mmol) was dissolved in diethyl ether (200 mL) and cooled to -80 ° C in a dry ice bath. Diisobutylaluminum hydride (32.0 mL, 32.0 mmol) was added to the reaction mixture and stirred for 25 minutes. The reaction mixture was removed from the dry ice bath and water (7.0 mL) was added. Ethyl acetate (200 mL) was added to the reaction mixture and stirred for 20 minutes. Magnesium sulfate (10 g) was added to the reaction mixture and stirred for 10 minutes. The reaction mixture was filtered through celite and concentrated to give a clear yellow oil (11.19 g). The yellow oil was chromatographed on silica, eluting with ethyl acetate and hexanes. The product (8.61, 87%) was a clear light yellow oil.

Example U-4) Triethyl phosphonoacetate (6.2 mL, 31.2 mmol) was dissolved in toluene (30 mL), placed in an ice bath under nitrogen and cooled to 0 ° C. To the reaction mixture, potassium bis (trimethylsilyl) amide (70 mL, 34.9 mmol) was added and stirred for 90 minutes. To the reaction mixture was added the product from Example U-3 (8.51 g, 24.6 mmol) dissolved in toluene (20 mL) and stirred for 1 hour. The reaction mixture was allowed to warm to room temperature. Potassium hydrogen sulfate (25 mL, 25 mmol) was added to the reaction mixture and stirred for 20 minutes. The mixture was extracted with ethyl acetate (3 × 100 mL), dried over magnesium sulphate and concentrated to give a pale brownish yellow oil (12.11 g). The oil was chromatographed on silica, eluting with ethyl acetate and toluene to give a light yellow oil (7.21 g, 70%).

Example U-5) The product from Example U-4 (5.0 g, 12.03 mmol) was dissolved in diethyl ether (100 mL) and placed in a dry ice bath and cooled to -80 ° C. Diisobutylaluminum hydride (21.0 mL, 21.0 mmol) was added to the reaction mixture. Stir for 30 minutes. Water (10 mL) was added to the reaction mixture, removed from the dry ice bath and stirred for 60 minutes. Magnesium sulfate (10 g) was added to the reaction mixture and stirred for 10 minutes. The reaction mixture was filtered over celite and concentrated to give a yellow oil (5.0 g). The oil was chromatographed on silica, eluting with ethyl acetate and hexanes to give a light yellow oil (2.14 g, 47%).

Example U-6) The product from Example U-5 was dissolved in tetrahydrofuran (50 mL). To the reaction mixture was added triphenyl phosphine (3.00 g, 8.84 mmol) on the polymer, oxadiazolinone (720 mg, 7.23 mmol), and azodicarboxylic acid dimethyl ester (1.17 g, 3.21 mmol) and room temperature for 6 hours. Stirred at. The reaction mixture was filtered over celite and concentrated to give a cloudy yellow oil (2.81 g). The oil was chromatographed on silica, eluting with ethyl acetate in hexanes to give a colorless transparent oil (1.66 g, 68%).

Example U-7) The product from Example U-6 (300 mg, 0.66 mmol) was dissolved in a solution of acetic acid and water containing zinc metal (10 mL, 25/75) and sonicated for 3 hours. The reaction mixture was filtered over celite and chromatographed in reverse phase HPLC to give a colorless transparent residue (13 mg, 4%).

Example U) The product from Example U-7 (13.0 mg, 0.031 mmol) was dissolved in 2N HCl (1.22 mL, 2.44 mmol) and refluxed for 1 hour. The reaction mixture was cooled and concentrated to give a colorless clear oil (6.6 mg, 95%).

Example V:

(α, 2S) -α-aminohexahydro-7-imino-1H-azepine-2-hexanoic acid, trihydrate hydrochloride

Example V-1)

A three neck 3 L flask was purged with nitrogen and then charged with cyclohexanone (1.27 moles, 132 ml) and 500 ml of toluene. The stirred mixture was cooled to 0 ° C. and 157.2 g (1.1 equiv) of potassium t-butoxide were added. After stirring the mixture for 1 hour, the reaction mixture was mechanically stirred for 1 hour after a solution of 5-pentenyl bromide (1.27 mol, 136 ml) in 100 ml of toluene was noted for color and texture changes. Dropped in The reaction mixture was warmed to 25 ° C. and stirred overnight. It was then diluted with 800 mL of 1N KHSO ₄ , the organic phase was dried (MgSO ₄ ), filtered and evaporated to dryness to give 208.5 g of crude product. This material was then purified by vacuum distillation (under water aspirator pressure) to afford the title compound in 47% yield.

Example V-2)

The product of Example V-1 (93.67 g, 0.563 mol) was combined with EtOH (600 ml), water (300 ml), NaOAc (101.67 g, 1.24 mol), and NH ₂ OH.HCl (78.31 g, 1.13 mol). Combined in three neck 3 L flasks. The stirred reaction mixture was refluxed for 16 hours and then stirred at 25 ° C. for another 24 hours. All solvents were removed under reduced pressure and the residue was partitioned between diethyl ether (Et ₂ O, 500 mL) and water (200 mL). The aqueous layer was extracted with 3 X 200 mL ether. The combined organic layers were dried over MgSO ₄ , filtered and stripped in vacuo to give the title oxime (121.3 g, 100% crude yield).

Example V-3)

A three neck 3 L flask was purged with nitrogen and then charged with hexamethyldisiloxane (471.7 ml, 2.2 mol), toluene (500 ml), and phosphorus pentoxide (203.88 g, 1.4 mol). This heterogeneous mixture was refluxed (about 1.5 hours) until a clear solution was obtained. After the mixture was cooled to room temperature, the oxime product of Example V-1 (102.1 g, 0.563 mol) in 200 mL of toluene was added at 25 ° C. over 1 hour. The reaction product was again stirred for 4-6 hours (check by TLC: 50% EA in Hex, I ₂ ) and poured into ice water with thorough mixing. 250 g of NaCl was added to this ice slurry mixture and solid potassium carbonate was added to the resulting mixture to adjust the pH to 5. The slurry was extracted with 3 × 500 mL of diethyl ether (Et ₂ O) and the combined organic fractions were dried over MgSO ₄ , filtered and stripped in vacuo to afford a crude mixture of regioisomer lactam (84.6 g). It was.

Example V-4)

The product of Example V-3 was then chromatographed (silica: acetonitrile) for purification and regioisomeric separation. From the crude sample, the 7-pentenyl regioisomer was isolated in 50% yield and after chiral chromatography, the desired single enantiomer was isolated in 43% yield each.

Example V-5)

R-isomer product of Example V-4 (102.1 g, 0.56 mol), dry THF (800 ml), DMAP (68.9 g, 0.56 mol), di-t-butyl dicarbonate (Boc ₂ 0, 99 g, 0.45 mol) ) Were combined in a 3 neck 3 L flask purged with argon. The reaction mixture was warmed to 70 ° C. within 30 minutes before further 52.8 g of Boc ₂ 0 and 200 ml of dry THF were added. After 30 minutes, another 32 g of Boc ₂ O was added and the mixture was stirred at 70 ° C. for 1 hour. Another 36 g of Boc ₂ O was added and the mixture was stirred for 1 h. The reaction mixture was cooled to room temperature and the THF stripped at 18 ° C. to 20 ° C. under reduced pressure. The precipitate was filtered off, washed with 100 ml of ethyl acetate (EA) and discarded (about 45 g). The EA filtrate was diluted with an additional 500 ml of EA and then washed with 500 ml of 1N KHSO _4, 500 ml of saturated aqueous NaHCO ₃ and 500 ml of brine and dried over anhydrous Na ₂ SO ₄ for 12 hours. This EA extract was then treated with 20 g of DARCO, filtered through celite covered with MgSO ₄ and concentrated in vacuo to yield 150 g of the title product as dark brown oil.

Example V-6)

A three neck 3 L flask containing the product of Example V-5 (150 g, 0.533) dissolved in ₃ L of CH ₂ Cl ₂ was cooled to -78 ° C. A stream of O ₃ was passed through the solution for 2.5 hours until the color of the reaction mixture turned blue. The argon was then bubbled through the solution maintained at -60 ° C to -70 ° C until the solution became clear and colorless (about 30 minutes). The reaction was then refluxed after addition of dimethylsulfide (DMS, 500 mL) and this reflux continued for 24 h. Another 100 ml of DMS was added and reflux continued for 12 h. Another 100 ml of DMS was added and reflux was continued for an additional 12 hours. The solvent and excess DMS were then stripped at 20 ° C. on a rotary evaporator. The resulting yellow oil obtained was diluted with 500 mL of deionized water and extracted with EA 3 × 300 mL. The EA layer was dried over anhydrous MgSO ₄ , treated with 20 g of DARCO, filtered through a thin layer of celite covered with anhydrous MgSO ₄ , and all solvents were stripped under reduced pressure to give 156 g of the crude title product as orange yellow oil.

Example V-7)

100 mL of CH ₂ Cl ₂ in a sample of N- (benzyloxycarbonyl) -alpha-phosphonoglycine trimethyl ester (160 g, 0.48 mol) dissolved in 1 L of dichloromethane (CH ₂ Cl ₂ ) and cooled to 0 ° C. A solution of DBU (110.29 g, 0.72 mol) in water was added. The colorless transparent reaction mixture was stirred for 1 hour at 0 ° C. to 6 ° C., then the Boc-aldehyde product (150 g, 0.53 mol) of Example V-6 in 600 mL of CH ₂ Cl _{2 was} charged at −5 ° C. to −1 ° C. Dropped at The reaction mixture was stirred at this temperature for 30 minutes and then slowly warmed to 10 ° C. in about 1 hour. The reaction mixture was washed with 1N KHSO ₄ (500 mL), saturated aqueous NaHCO ₃ (200 mL) and 50 aqueous NaCl (200 mL). The organic layer was then dried over anhydrous MgSO ₄ , treated with 40 g of DARCO, filtered through a thin layer of celite covered with anhydrous MgSO ₄ , and concentrated to give 258 g of the crude title product as a yellow oil. The material was purified by chromatography to give 130 g (55%) of the crude title product.

Example V-8)

To a solution of MeOH (1 L) of the product of Example V-7 (91.3 g, 0.19 mol) was added 2.5 g of S, S-Rh-DIPAMP catalyst followed by hydrogen. Hydrogenation was performed at 25 ° C. for 1.5 hours in a Parr apparatus. The reaction mixture was filtered through celite and concentrated to afford the crude title product (90 g, 98%) as a brown oil.

Example V-9

To a solution of the product of Example V-8 (90 g,) in 200 mL of glacial acetic acid was added 200 mL of 4N HCl in dioxane. After the reaction mixture was stirred at 25 ° C. for 20 minutes, all solvents were stripped at 40 ° C. under reduced pressure to give a reddish brown oil. This oily product was treated with 500 mL of water and extracted with 2 × 300 mL of dichloromethane. The combined organic layers were washed with saturated sodium bicarbonate solution (100 mL), dried over magnesium sulfate, filtered and stripped all solvents to afford the crude title product. Chromatography of this material gave 45 g (62%) of pure title product.

Example V-10)

To a sample of 45.0 g (0.115 mole) of the product of Example V-9 in 300 ml of dichloromethane purged with argon was added 23.0 g (0.121 mole) of triethyloxonium tetrafluoroborate. The mixture was stirred at 25 ° C. for 1 hour and then 150 ml of saturated aqueous sodium bicarbonate solution was added. The dichloromethane layer was separated, washed with 150 ml of 50% NaCl aqueous solution, dried over sodium sulfate, filtered through celite and concentrated at 25 ° C. to give a clear yellow oil, 47.0 g (97%) of the title product. .

Example V-11)

To 7.0 g (0.130 mole) of ammonium chloride in 500 ml of methanol was added 31.2 g of the title substance (45.0 g, 0.107 mole) of Example V-10. The reaction was refluxed at 65 ° C. for 5 hours and then all solvents were removed under reduced pressure to yield 40 g (87%) of crude product as foam viscous mass. This material was purified by column chromatography to give 37 g (81%) of the title product.

Example V)

The title product (36.0 g, 0.084 mol) of Example V-11 in 1 L of 2.3N HCl was refluxed for 3 hours. After cooling to room temperature, the solution was washed with 2x150 mL of CH ₂ Cl ₂ and then all solvents were stripped under vacuum to yield 25.6 g (96%) of the title amino acid product as a pale yellow foam.

Example W:

(αS, 2R) -α-aminohexahydro-7-imino-1H-azepine-2-hexanoic acid, trihydrate hydrochloride

Example W-1

The S-isomer product of Example V-4 (5.45 g, 0.030 mol) was converted to its Boc derivative by the method of Example V-5. After chromatography, the reaction yielded 6.3 g (75%) of the desired title product.

Example W-2

The product of Example W-1 (6.3 g, 0.025 mol) was ozonated by the method of Example V-6 to yield 8.03 g of crude title aldehyde which was used without further purification.

Example W-3)

The product of Example W-2 (8.03 g, 0.024 mol) was converted to N- (benzyloxycarbonyl) -alpha-phosphonoglycine trimethyl ester (7.9 g, 0.024 mol) using the procedure of Example V-7. Condensation gave 4.9 g (44%) of the desired title product after chromatography.

Example W-4)

The product of Example W-3 (4.8 g, 0.010 mol) was reduced in the presence of R, R-Rh-DIPAMP catalyst by the method of Example V-8 to give 2.9 g (60%) of the desired title product after chromatography. ) Was obtained.

Example W-5)

The product of Example W-4 (2.9 g, 0.006 mol) was deprotected by treatment with HCl using the method of Example V-9 to yield 2.3 g (100%) of the desired title product.

Example W-6)

The product of Example W-5 (0.56 g, 0.0015 mol) was alkylated with triethyloxonium tetrafluoroborate using the method of Example V-10 to yield 0.62 g (98%) of the desired title product.

Example W-7)

The product of Example W-6 (0.62 g, 0.0015 mol) was treated with ammonium chloride in methanol using the method of Example V-11 to give 0.50 g (88%) of the desired title product after chromatography purification. It was.

Example W-8)

The product of Example W-7 (0.37 g, 0.0009 mol) dissolved in MeOH was added to the Parr hydrogenation apparatus. 5% Pd / C of catalytic amount was added to this vessel. Hydrogen was introduced and the reaction was carried out over a period of 7 hours at a pressure of 5 psi at room temperature. The catalyst was removed by filtration and all solvents were removed from the filtrate under reduced pressure to yield 0.26 g (quantitative) of the desired title product.

Example W)

The solution of the product of Example W-8 dissolved in 2N HCl (30 mL) was kept under reflux for 2 hours and then cooled to room temperature. All solvents were removed under reduced pressure and the residue was dissolved in 50 mL of water. Again in this solution all the solvents were stripped under reduced pressure and then dissolved again in 12 ml of water and then lyophilized to yield 0.245 g (71%) of the title compound.

Example X:

(αS, 2S) -α-aminohexahydro-7-imino-1H-azepine-2-hexanoic acid, trihydrate hydrochloride

Example X-1)

Cyclohexanone (4500.0 g, 45.85 moles), acetone dimethyl acetal (5252.6 g, 50.43 moles) in a 22 L round bottom flask equipped with an overhead stirrer, half moon paddle, heating mantle, thermocouple and silver vacuum jacketed distillation column (5 plates) ), Allyl alcohol (6390.87 g, 110.04 mol) and p-toluene sulfonic acid (PTSA) (0.256 g, 0.001 mol). After starting agitation (137 rpm) the vessel was slowly heated with the initial set point to 70 ° C. The heating was stepwise increased to a final vessel temperature of 150 ° C. The determination of whether to increase the reactor set point was made based on the distillation rate. If the distillation rate slowed or stopped, additional heating was applied. Additional heating to 150 ° C. resulted in Claisen rearrangement. After the vessel temperature rose to 150 ° C. and no distillation was observed, the heating mantle was lowered and the reaction mixture was cooled to 130 ° C. PTSA was then neutralized with 3 drops of 2.5N NaOH. The heating mantle was then lowered off the flask and the vacuum stripping started. Evaporative cooling was used to lower the vessel temperature and to gradually lower the pressure to 40 mmHg. When the vessel temperature was reduced to about 100 ° C., the heating mantle was raised back to the proper position for heating. Unreacted cyclohexanone and low boiling impurities were distilled off. The vessel temperature was slowly raised (the maximum temperature difference between the vessel and the steam was about 12 ° C). The product was isolated at 109-112 ° C. at 40 mmHg. Typical yields were 40 to 45%. Fractions were combined and redistilled to a less than 95% area (GC) to give the title product in 55% total yield.

Example X-2)

Add hydroxyl amine-O-sulfonic acid (91.8 g) dissolved in acetic acid (470 g) to a 1 L Bayer flask equipped with a mechanical stirrer, thermocouple, condenser cooled to 0 ° C. and an addition funnel and heated to 70 ° C. It was. Allyl cyclohexone (100 g) was added dropwise to the solution in about 40 minutes while maintaining the temperature at 70-78 ° C. During the addition, the reaction appearance changed from a white slurry to a clear orange solution. After addition, the reaction was heated and stirred at 75 ° C. for a further 5 hours. IPC samples were taken each hour. After the reaction was completed, acetic acid was stripped in a rotary evaporator at 50 ° C. under reduced pressure. Water (200 mL) was then added to the residue and the solution extracted with toluene (2 X 300 mL). The organic layers were combined, treated with water (150 mL) and stirred for 10 minutes. Sodium hydroxide solution (79.4 g of 50 solution) was added until the aqueous layer became basic (pH 12). Neutralization was carried out by controlling the temperature below 40 ° C. in the reactor. The layers were then separated and the toluene layer was passed through a filter to remove any solid or tar material. The organic solution was then stripped in a rotary evaporator at 50 ° C. under reduced pressure. The residue was taken up in a mixture of toluene (510 mL) and heptane (2040 mL) and heated to 60 ° C. in a 3 L beaker. A clear yellow-orange solution was obtained. The title product begins to crystallize at 53 ° C. as the solution is slowly cooled to 5 ° C. with stirring. The solid was filtered off, washed with heptane (50 mL) and evaporated at 40 ° C. under house vacuum overnight to give 66.3 g (60%) of the title product as greyish white crystals. A portion of this material was recrystallized from toluene and heptane to yield the title product as a white crystalline solid.

Example X-3)

The racemic product mixture of Example X-2 was separated by chiral chromatography eluting with 100% acetonitrile on a Chiralpac AS 20 um column. 220 nM wavelength was employed in the detector. Sample loads of acetonitrile 0.08 g / mL were used to obtain 90% recovery of the isomers, each of which was greater than 95% ee. A portion of the R-isomer material was recrystallized from toluene and heptane to produce the R-isomer title product as a white crystalline solid.

R-isomer: Melting point = 81 to 82 ° C.

Example X-4)

A five neck flat bottom flask equipped with a dropping funnel, thermometer and mechanical overhead stirrer was emptied and purged three times with nitrogen. The R-isomer product lactam of Example X-3 (100.0 g, 0.653 mol), DMAP (7.98 g, 65 mmol) and N-diisopropylethyl amine (Hunig base, 113.3 g, 0.876 mol) were added to toluene (350 ML) and di-t-butyl dicarbonate (170.2 g, 0.78 mol) dissolved in toluene (100 mL) were added. (Note: the reaction is better when using 2.0 equivalents of Hunig's base). The mixture was heated to 65 ° C. (Note: stable degassing was observed during the reaction). After 1.5 h, another 86.25 g of di-t-butyl-dicarbonate (0.395 mol) dissolved in toluene (50 mL) was added. Heating was continued for 17 hours and IPC by HPLC showed 75 conversions. Another 42.78 g of di-t-butyl dicarbonate (0.196 mol) in toluene (30 mL) was added and the brown mixture was heated for 5.5 h. After cooling to ambient temperature, the mixture was treated with 4M HCl (215 mL) and the aqueous layer was extracted with toluene (2x80 mL). The combined organic layers were washed with NaHC0 ₃ (170 mL) and 250 mL of water (note: the internal temperature during the quenching process is controlled by external cooling with ice / water). Gas evolution was observed. The organic layer was evaporated to give 257.4 g of a brown liquid. This crude material was purified by plug filtration over SiO ₂ (950 g) using toluene / EtOAc 9/1 (6 L) and toluene / AcOEt 1/1 (0.5 L) as eluent to afford 139.5 g (51) of a yellow liquid title product. %) Was obtained.

Example X-5)

Example X-6)

Example 1f

2-h stainless steel autoclave equipped with baffle and 6-blade gas dispersion shaft impeller Rh (CO) ₂ (acac) (0.248 g, 0.959 mmol), BIPHEPHOS And prepared as described in Example 13 of US Pat. No. 4,769,498, 2.265 g, 2.879 mmol), the product of Example X-4 (N- (t-butoxycarbonyl) -S-7-allyl Caprolactam (242.9 g, 0.959 mole), and toluene (965 g) were charged The reactor was sealed and purged with 100% carbon monoxide (8 x 515 kPa) The reactor was pressurized to 308 kPa (30 psig) with 100% carbon monoxide Then, a total pressure of 515 kPa (60 psig) was achieved by adding a 1: 1 CO / H ₂ gas mixture, while adding the mixture to maintain a total pressure of about 515 kPa (60 psig) while vigorously mechanically shaking. Heated together with a 1: 1 CO / H ₂ gas mixture which was then heated to 50 ° C. After 22 hours, the mixture was cooled to about 25 ° C. and the pressure was carefully released The product mixture was vacuum filtered and the filtrate was evaporated under reduced pressure. 267.7 g of a light yellow oil were obtained The analysis by ¹ H NMR showed the corresponding aldehyde production of Example V-6. This was consistent with the inherent quantitative conversion of starting material with about 96% selectivity to water The oil was used in the following examples without further purification.

Example X-8)

Example 1 g

To a sample of N- (benzyloxycarbonyl) -alpha-phosphonoglycine trimethyl ester (901.8 g, 2.7 mol) dissolved in CH ₂ Cl ₂ and cooled to 0 ° C., DBU in CH ₂ Cl ₂ (597.7 g, 3.9) Mole) was added. The colorless transparent reaction mixture was stirred for 1 hour at 0 ° C. to 6 ° C. and then a sample of the Boc-aldehyde product (812.0 g, 2.9 mol) of Example V-6 in CH ₂ Cl ₂ was taken from -5 ° C. to -1 ° C. Dropped at The reaction, workup and purification were completed as described in Example V-7 to afford 1550 g of the title product of Example V-7 containing a small amount of CH ₂ Cl ₂ .

Example X-9

To a solution of MeOH (1 L) of the product of Example V-7 (100 g, 0.20 mol) was added 3 g of RR-Rh-DIPAMP catalyst. Hydrogenation was performed in a Parr apparatus at 25 ° C. for 1.5 hours. The reaction mixture was filtered through celite and then concentrated to afford the title product of crude Example X-9 as brown oil (100 g).

Example X-10)

To a solution of the product of Example V-8 (100 g) in 200 mL glacial acetic acid was added 25 mL 4N HCl in dioxane. The reaction mixture was stirred at 25 ° C. for 20 minutes and then stripped all solvents at 40 ° C. under reduced pressure to yield 105 g of reddish brown oil. This oily product was treated with 500 mL of water and extracted with 2 × 300 mL of dichloromethane. The combined organic layers were washed with saturated sodium bicarbonate solution (100 mL), dried over magnesium sulfate, filtered and stripped all solvents to afford 99.9 g of the title product as reddish brown oil.

Example X-11)

600 mL of dichloromethane purged with argon To 10.0 g (0.077 mol) of the product of Example X-10 was added 15.7 g (0.082 mol) of triethyloxonium tetrafluoroborate. The mixture was stirred for 1 h at 25 ° C., then 300 ml of saturated aqueous sodium bicarbonate solution were added. The dichloromethane layer was separated, washed with 300 mL of 50% NaCl aqueous solution, dried over sodium sulfate, filtered through celite and concentrated at 25 ° C. to give 31.2 g (-97%) of a clear yellow oil of the title product. .

Example X-12)

To 4.2 g (0.078 mole) of ammonium chloride in 500 ml of methanol was added 31.2 g of the titled material of Example X-11. The reaction was refluxed at 65 ° C. for 5 hours and then all solvents were removed under reduced pressure to yield 29 g (92%) of crude product as a foamy viscous mass. This material was purified by column chromatography to give 23 g (70%) of the title product.

Example X)

The title product (23 g) of Example X-12 in 500 mL of 2N HCl was refluxed for 5 hours. Then all solvents were removed in vacuo and the residue redissolved in water was washed with ₂ × 300 mL of CH ₂ Cl ₂ . The aqueous layer was then concentrated in vacuo to yield 17 g (100%) of a light brown hygroscopic solid title product.

Example Y

(αR, 2S) -α-aminohexahydro-7-imino-1H-azepine-2-hexanoic acid, trihydrate hydrochloride

Example Y-1)

A solution of Example X-3 (3.0 g, 0.015 mol) in methylene chloride and methanol (75/45 mL) was cooled to -78 ° C in a dry ice bath. The reaction was stirred while bubbling with ozone at a flow rate of 3 ml / min through the solution. If the solution remained a consistent dark blue color, ozone was removed and the reaction was purged with nitrogen. Sodium borohydride (2.14 g, .061 mol) was added very slowly to the cold solution to minimize gas evolution at one time. Glacial acetic acid was added slowly to the reaction to pH 3. The reaction was then neutralized with saturated sodium bicarbonate. The organics were then washed with 3x 50 mL of brine, dried over magnesium sulfate anhydride and removed under reduced pressure. The light oil was passed through a plug of silica (15 g) to afford 5.15 g, 0.026 mol (64%) of alcohol. C ₉ H ₁₄ N ₂ O ₃ .

Example Y-2)

To the solution of Example Y-1 (5.15 g, 0.026 mol) in methylene chloride (100 mL) was added carbon tetrabromide (10.78 g, 0.033 mol) in an ice bath at 0 ° C. The solution was cooled to 0 ° C. in an ice bath. Thereafter, triphenylphosphine (10.23 g, 0.39 mol) was divided into portions to prevent the temperature from rising above 3 ° C. The reaction was stirred for 2 hours and the solvent was removed in vacuo. The crude material was purified by flash chromatography to give bromide (5.9 g, 0.023 mole) in 87% yield.

Example Y-3)

Triphenyl phosphine (7.17 g, 0.027 mol) was added to a solution of Example Y-2 (5.71 g, 0.026 mol) in toluene (25 mL). The reaction was refluxed for 16 h in an oil bath. After cooling, toluene was decanted from the glassy solid. The solid was triturated with diethyl ether overnight to give phosphonium bromide (10.21 g, 0.020 mol) in 90% yield.

Example Y-4)

To a 1 L round bottom flask was added N-benzyloxycarbonyl-D-homoserine lactone (97 g, 0.442 mol) in ethanol (500 mL). To the reaction was added a solution of sodium hydroxide (1M, 50 mL). The reaction was monitored by thin layer chromatography until the starting material was consumed for 12 hours. Toluene (60 mL) was added and then the solvent was removed in vacuo. The residue was used without further purification.

Example Y-5)

The residue from Example Y-4 was suspended in DMF in a 1 L round bottomed flask. Benzyl bromide (76.9 g, 0.45 mol, 53.5 mL) was added to the suspension and the mixture was stirred for 1 hour. Samples were quenched and analyzed by mass spectroscopy to confirm the consumption of starting material and no lactone reformation. To the reaction was added 1 L of ethyl acetate and 500 mL of brine. The aqueous layer was washed twice more with 500 ml of ethyl acetate. The organics were combined, dried over MgSO ₄ and concentrated. Silica gel chromatography gave N-benzyloxycarbonyl-S-homoserine benzyl ester as a white solid (80 g).

Example Y-6)

To a 2 L round bottom flask was added pyridinium chlorochromate (187 g, 0.867 mole) and silica gel (197 g) suspended in CH ₂ Cl ₂ (600 mL). To the slurry was added a solution of the product of Example Y-5 (80 g, 0.233 mol) in CH ₂ Cl ₂ (600 mL). The mixture was stirred for 4 hours. Thin layer chromatography showed that the starting material was consumed. 1 L of diethyl ether was added to the reaction. The solution was then filtered through a pad of celite and then through a pad of silica gel. The solvent was removed in vacuo and the resulting oil was purified by silica gel chromatography to give aldehyde (58.8 g) in 38% overall yield.

Example Y-7)

To a 3L three neck flask was added phosphonium salt (56.86 g, 0.11 mol) from Example Y-3 in THF (1 L) dried under vacuum on P ₂ O ₅ . The slurry was cooled to -78 ° C in a dry ice bath. KHMDS (220 mL, 0.22 mol) was added dropwise to the cold slurry so that the temperature did not rise above -72 ° C. The reaction was stirred at -78 ° C for 20 minutes and then at -45 ° C for 2 hours. Thereafter, the temperature was lowered to −78 ° C., and aldehyde (15.9 g, 0.047 mol) from Example Y-6 was added dropwise in THF (50 mL) over 45 minutes. The reaction was stirred at −77 ° C. for 30 minutes, then warmed to −50 ° C. for 1 hour and then to room temperature over 4 hours. To the reaction was added ethyl acetate (200 mL) and saturated ammonium chloride. The organics were collected, dried over MgSO ₄ and concentrated in vacuo. The crude oil was purified by silica chromatography to give the olefin compound (45.1 g) as a pale yellow viscous oil in 81% yield.

Example Y)

To a 20 mL glass jar was added the product from Example Y-7 (19.77 g, 0.039 mol) in dioxane (50 mL) and 4N aqueous HCl (250 mL). To this solution was added a catalytic amount of 10% Pd on carbon in a hydrogenation flask. The flask was pressurized with H ₂ (50 psi) for 5 hours. The reaction was monitored by mass spectroscopy and the starting material was consumed. The solution was filtered through a pad of celite and washed with water. Solvent was removed by lyophilization to give the title compound (7.52 g) in 81% yield.

Example Z

(αS, 2S) -α-aminohexahydro-7-imino-1H-azepine-2-hexanoic acid, trihydrate hydrochloride

Example Z-1)

To a 1 L three neck flask was added phosphonium salt (21.21 g, 0.041 mol) from Example Y-3 in THF (200 mL). The slurry was cooled to -78 ° C in a dry ice bath. KHMDS (88 mL, 0.044 mol) was added dropwise to the cold slurry so that the internal temperature did not rise above -72 ° C. The reaction was stirred at −78 ° C. for 20 minutes and then at −45 ° C. for 1 hour. The temperature was then lowered back to −78 ° C. and aldehyde (15.9 g, 0.047 mol) (prepared as in Example Y (4-6) using N-benzyloxycarbonyl-L-homoserine lactone) It was added dropwise in THF (50 mL) over 45 minutes. The reaction was stirred at −77 ° C. for 30 minutes, then warmed to −50 ° C. for 30 minutes and then to room temperature over 4 hours. To the reaction was added ethyl acetate (100 mL) and saturated ammonium chloride. The organics were collected, dried over MgSO ₄ and concentrated in vacuo. The crude oil was purified on silica chromatography to give the olefin compound (9.0 g) as a pale yellow viscous oil in 45% yield.

Example Z)

To the vial was added the product from Example Z-1 in dioxane (5 mL) and 4N aqueous HCl (16 mL). To this solution a catalytic amount of 10% Pd on carbon was added in a hydrogenation flask. The flask was pressurized with H ₂ (50 psi) for 5 hours. The reaction was monitored by mass spectroscopy and the starting material was consumed. The solution was filtered through a pad of celite and washed with water. Solvent was removed by lyophilization to give the title compound (98.7 mg) in 79.4% yield.

Example AA

(2S, 4Z) -2-Amino-6-[(2R) -hexahydro-7-imino-1H-azin-2-yl] -4-hexenoic acid

Example AA-1)

(2S, 4Z) -6-[(2R) -hexahydro-7-imino-1H-azin-2-yl] -2-[[(phenylmethoxy) carbonyl] amino] -4-hexenoic acid , Phenylmethyl ester

To a 50 mL flask was added a sample of Example Z-1 (1.5 g, 2.97 mmol) in methanol (25 mL). Thereafter, a 60% solution (16 mL) of glacial acetic acid was added to the reaction mixture. A precipitate was observed. Additional methanol was added to dissolve the solids (1 mL). Thereafter, zinc powder was added to the reaction (0.200 g). The reaction was sonicated for 4 hours while the temperature was maintained at 37 ° C. The reaction was monitored by TLC and MS until the starting material was consumed and the mass corresponding to the product was observed. The solution was decanted from zinc and acetonitrile / water 30% solution (100 mL) was added to the filtrate. The reaction was purified with 52% acetonitrile / water [gradient from 20% to 70% acetonitrile over 30 minutes] in two runs on Waters preparative HPLC. The resulting product was lyophilized to give the title material (1.01 g) of Example AA-1 in 73% yield as a white solid.

Example AA)

To a 250 mL flask was added the product of Example AA-1 (1.0 g, 2.2 mmol) in 4M HCl (100 mL). The reaction was refluxed overnight and monitored by MS until the starting material was consumed and mass on the product was observed. The reaction was purified using 18% acetonitrile / water [from 0% to 30% acetonitrile / water over 30 minutes] in two runs on a Waters preliminary reversed phase column without further finishing. The combined fractions were lyophilized to give the title product (0.34 g) as a cream colored foam in 64% yield.

Example BB

(2S, 4E) -2-Amino-6-[(2R) -hexahydro-7-imino-1H-azin-2-yl] -4-hexenoic acid

Example BB-1)

(2S, 4E) -2-[[(phenylmethoxy) carbonyl] amino] -6-[(5R) -6,7,8,9-tetrahydro-3-oxo-3H, 5H- [1, 2,4] oxadiazolo [4,3-a] azin-5-yl] -4-hexenoic acid, phenylmethyl ester

To a 250 mL flask was added Example Z-1 (2.0 g, 3.9 mmol) and phenyl disulfide (0.860 g, 3.9 mmol) in a cyclohexane (70 mL) / benzene (40 mL) solution. Nitrogen was bubbled through the solution to purge the oxygen of the system. The reaction was exposed to short wavelength UV lamp for the weekend. The reaction was evaluated by normal HPLC (ethyl acetate / hexane). 71% trans isomer and 29% cis isomer were observed. Exposure of the reaction to UV for an additional 3 days converted 84% of the starting material to trans isomer and 16% of the starting cis isomer remained. Purification by chromatography gave Example BB-1 (0.956 g) in 48% yield.

Example BB-2)

(2S, 4E) -6-[(2R) -hexahydro-7-imino-1H-azin-2-yl] -2-[[(phenylmethoxy) carbonyl] amino] -4-hexenoic acid , Phenylmethyl ester, monohydrochloride

Sample (0.956 g, 1.9 mmol) of the product of Example BB-1 in MeOH (80 mL) was purified by Zn powder (1.5 g) and 60% HOAc / H ₂ O (40 mL) by the method of Example AA-1. Deprotection. The resulting product was purified by reverse phase chromatography to give the title material (0.248 g) in 28% yield.

Example BB)

The product of Example BB-2 (0.248 g, 0.53 mmol) was purified by HCl (2 mL), H ₂ O (2 mL), CH ₃ CN (4 mL) by the method of Example AA. It was transformed into. The crude product was purified by reverse phase chromatography to give the title product (0.073 g) of Example BB in 57% yield.

Example CC

(E) -2-amino-2-methyl-6-[(1-iminoethyl) amino] -4-hexenoic acid, dihydrochloride

Example CC-1)

DL-alanine ethyl ester hydrochloride (5 g, 32.5 mmol) was suspended in toluene (50 mL). Triethyl amine (4.5 mL, 32.5 mmol) was added followed by phthalic anhydride (4.8 g, 32.5 mL). The reaction flask was retrofitted with a Dean-Stark trap and reflux condenser, and the mixture was heated at reflux overnight. About 10 ml of toluene / water was collected. The reaction mixture was cooled to rt and diluted with aqueous NH ₄ Cl and EtOAc. The layers were separated and the aqueous layer was extracted with EtOAc (3X). The ethyl acetate extract was washed with brine, dried over MgSO ₄ , filtered and concentrated in vacuo to give the title phthalyl-protected amino ester as a white crystalline solid in near quantitative yield.

Example CC-2)

Potassium phthalimide (18.5 g, 0.1 mol) was added to a 250 ml round bottom flask containing 1,4-butene-dichloride (25 g, 0.2 mol). The reaction mixture was heated to 150 ° C. for 1.5 h. The mixture was cooled to room temperature and layered between brine and Et ₂ O. The organic layer was dried over MgSO ₄ , filtered and concentrated in vacuo. The residue was recrystallized from hot ethanol to give the title 1-chloro-4-phthalimidobutene (8.9 g, 39%) as orange crystals.

Example CC-3)

A sample of the product of Example CC-2 (2.3 g, 9.8 mmol) was dissolved in acetone (50 mL). NaI (3.2 g, 21 mmol) was added and the mixture was refluxed overnight. After cooling to room temperature, Et ₂ O was added and the mixture was washed sequentially with sodium thiosulfate and brine. The organic layer was dried over MgSO ₄ , filtered and concentrated in vacuo to yield the title iodide (2.8 g, 87.5%) as a light yellow solid, which was used without further purification.

Example CC-4)

A solution of KHMDS (2.6 g, 13.3 mmol) in THF (50 mL) was cooled to -78 ° C. A solution of the product of Example CC-1 (2.2 g, 8.87 mmol) in THF (15 mL) was added and 1,3-dimethyl-3,4,5,6-tetrahydro-2 (1H) -pyrimi Dinone (DMPU, 1.0 mL, 8.87 mL) was added immediately thereafter. The solution was stirred at −78 ° C. for 40 minutes before the solution of the product of Example CC-3 (2.9 g, 8.87 mmol) in THF (15 mL). The flask was removed from the cold bath and stirred at room temperature for 3 hours. The reaction mixture was partitioned between saturated aqueous NaHCO ₃ and EtOAc. The organic extract was washed with brine, dried over MgSO ₄ , filtered and concentrated in vacuo to afford the desired bis-phthalyl protected amino ester as a yellow solid. This residue was chromatographed on silica gel (1: 1 hexanes: EtOAc) to give 1.4 g (35%) of the title material as a white solid.

Example CC-5)

The product of Example CC-4 (0.78 g, 1.76 mmol) was dissolved in a mixture of formic acid (10 mL, 95%) and HCl (20 mL, concentrated HCl) and refluxed for 3 days. The reaction mixture was cooled to 0 ° C. and filtered to remove phthalic anhydride. After concentration in vacuo (T <40 ° C.), the title unsaturated alpha methyl lysine was obtained as a white solid (0.38 g, 95%), which was used without further purification.

Example CC)

The product of Example CC-5 (0.2 g, 0.86 mmol) was dissolved in H ₂ O (8 mL) and brought to pH 9 with 2.5 N NaOH. Ethyl acetamidate-HCl (0.42 g, 3.4 mmol) was added in portions over 1 hour. After 1 h, the mixture was acidified to 10% HCl to pH 4 and concentrated in vacuo. The residue was then passed through a water-washed DOWEX 50WX4-200 column (H form, 0.5 N NH ₄ OH eluate). The residue was concentrated in vacuo, acidified with 10% HCl to pH 4 and concentrated to give the title product as an oil (17 mg, 6%).

Example DD

(R, E) -2-Amino-2-methyl-6-[(1-iminoethyl) amino] -4-hexenoic acid, dihydrochloride

Example DD-1)

(2S, 4S) -3-benzoyl-2- (t-butyl) -4-methyl-1,3-oxazolidin-5-one was prepared according to Seebach's procedure. Seebach, D .; Fadel, A., "Helvetica Chimica Acta 1985,68, 1243".

Example DD-2)

A solution of KHMDS (0.65 g, 3.24 mmol), DMPU (0.33 mL, 2.7 mmol) and THF (40 mL) was cooled to -78 ° C. (2S, 4S) -3-benzoyl-2- (t-butyl) -4-methyl-1,3-oxazolidin-5-one (Example DD-1) (0.70 g, in THF (10 mL) 2.7 mmol) was added dropwise. After 45 minutes, a solution of the product of Example CC-3 (0.88 g, 2.7 mmol) in THF (10 mL) was added. The reaction mixture was stirred at rt for 2 h and quenched with saturated aqueous NaHCO ₃ . The layers were separated and the aqueous layer was extracted with EtOAc. The organic layers were combined, washed with brine, dried over MgSO ₄ , filtered and concentrated in vacuo. Chromatography of the resulting yellow oil on silica gel (9: 1, then 4: 1 hexanes / ethyl acetate) gave the titled protected unsaturated alpha methyl D-lysine (0.26 g, 20%) as a colorless oil. Obtained.

Example DD-3)

The product of Example DD-2 (0.255 mg, 0.55 mmol) was dissolved in 6N HCl (6 mL) and formic acid (6 mL) and heated to reflux for 24 h. The reaction mixture was cooled to rt and concentrated in vacuo. The residue was suspended in water and washed with CH ₂ Cl ₂ . The aqueous layer was concentrated and passed through a water-washed DOWEX 50WX4-200 column (H form, 0.5 N NH ₄ OH eluate). The residue was concentrated in vacuo, acidified to 10% HCl to pH 4 and concentrated to give the title unsaturated D-lysine (71 mg, 55%) as an oil, which was used without further purification.

Example DD)

The product of Example DD-3 (13 mg, 0.056 mmol) was dissolved in H ₂ O (5 mL) and brought to pH 9 with 2.5 N NaOH. Ethyl acetamidate-HCl (27 mg, 0.2 mmol) was added in 4 portions over 2 hours. After 2 hours, the mixture was acidified with 10% HCl to pH 4 and concentrated in vacuo. The residue was passed through a water-washed DOWEX 50WX4-200 column (H form, 0.5 N NH ₄ OH eluate). The residue was concentrated in vacuo, acidified with 10% HCl to pH 4 and concentrated to give the title product as an oil (45 mg).

Example EE

(S, E) -2-amino-2-methyl-6-[(1-iminoethyl) amino] -4-hexenoic acid, dihydrochloride

Example EE-1)

(2R, 4R) -3-benzoyl-2- (t-butyl) -4-methyl-1,3-oxazolidin-5-one was prepared according to Seebach's procedure. Seebach; Padel, "Helvetica Chimica Acta 1985,68, 1243".

Example EE-2)

(2R, 4R) -3-benzoyl-2- (t-butyl) -4-methyl-1,3-oxazolidin-5-one product of Example EE-1 in THF (50 mL) (2.0 g, 7.6 mmol) was cooled to -78 ° C. A -78 ° C solution of KHMDS (0.65 g, 3.24 mmol) in THF (25 mL) was added dropwise. After 30 minutes, a solution of the product of Example CC-3 (2.8 g, 8.6 mmol) in THF (25 mL) was added. The reaction mixture was stirred for 1 h under room temperature and quenched with saturated aqueous NaHCO ₃ . The layers were separated and the aqueous layer was extracted with EtOAc. The organic layers were combined, washed with brine, dried using MgSO ₄ , filtered and concentrated in vacuo. The resulting orange oil was chromatographed on silica gel (9: 1, then 4: 1 hexanes / ethyl acetate) to give the titled unsaturated alpha methyl L-lysine (0.5 g, 15%) as a white solid.

Example EE-3)

The product of Example EE-2 (0.5 g, 1 mmol) was dissolved in 12N HCl (10 mL) and formic acid (5 mL) and the mixture was heated to reflux for 12 h. The reaction mixture was cooled in the freezer for 3 hours and the solids were removed by filtration. The residue was washed with CH ₂ Cl ₂ and EtOAc. The aqueous layer was concentrated in vacuo to yield the title unsaturated alpha methyl L-lysine (0.26 g, 99%) as an oil, which was used without further purification.

Example EE)

The product of Example EE-3 (0.13 g, 0.56 mmol) was dissolved in H ₂ O (1 mL) and brought to pH 9 with 2.5 N NaOH. Ethyl acetamidate-HCl (0.28 g, 2.2 mmol) was added in four portions over 1 hour. After 1 h, the mixture was acidified with 10% HCl to pH 4 and concentrated in vacuo. The residue was passed through a water-washed DOWEX 50WX4-200 column (0.5 N NH ₄ OH eluate). The residue was concentrated in vacuo, acidified with 10% HCl to pH 4 and concentrated to give the title product as an oil (40 mg).

Example FF

2-Amino-2-methyl-6-[(1-iminoethyl) amino] -4-hexynoic acid, dihydrochloride

Example FF-1)

N-boc-1-amino-4-chlorobut-2-yne was prepared following the procedure described in Tetrahedron Lett. 21,4263 (1980).

Example FF-2)

Methyl N- (diphenylmethylene) -L-alanineate was prepared following the procedure described in J. Org. Chem., 47,2663 (1982).

Example FF-3)

Anhydrous THF (1000 mL) was placed in an argon-purged flask and 60% NaH (9.04 g, 0.227 mol) dispersed in mineral oil was added. To this mixture was added the product of Example FF-2 (30.7 g, 0.114 mol). The reaction mixture was then stirred at 10-15 ° C. for 30 minutes. Potassium iodide (4 g) and iodine (2 g) were added, and within 30 minutes the product of Example FF-2 (23 g, 0.113 mole in 200 mL of THF) was added. The reaction mixture was then stirred at 55 ° C. until the starting material disappeared (about 2 hours). Then the reaction mixture was cooled to room temperature and the solvent was evaporated. Ethyl acetate (500 mL) was added and the mixture was washed carefully with deionized water (2 × 200 mL). The organic layer was dried over anhydrous MgSO ₄ , filtered and evaporated to give 44 g of crude product. Purification by chromatography using 20% ethyl acetate in hexanes gave the title protected unsaturated alpha-methyl lysine (28 g, 57%).

Example FF-4)

The product of Example FF-3 (16 g, 0.0368 mol) was dissolved in 1N HCl (300 mL) and stirred at 25 ° C for 2 h. The reaction mixture was washed with ether (2 x 150 mL), the aqueous layer was separated and decolorized with charcoal. Concentration yielded about 9 g (100% yield) of deprotected unsaturated alpha-methyl lysine ester (FF-4) as a white herb solid.

Example FF-5)

The product of Example FF-4 (2.43 g, 0.01 mol) was dissolved in deionized water (25 mL). A solution of NaOH (400 mg, 0.01 mol) in deionized water (25 mL) was added at 25 ° C. to bring the pH to about 7.95, followed by further stirring for 10 minutes. 1N NaOH was added to adjust the pH to about 8.5, while ethyl acetamidate hydrochloride (988 mg, 0.008 mol) was added to the reaction mixture. After addition of acetimidate, the reaction mixture was stirred at pH 8-8.5 for 3 hours. 1 N HCl was added to the reaction mixture (4.1 pH). The solvent was evaporated at 50 ° C. to yield a crude yellow hygroscopic residue (4 g,> 100% yield). Purification on a Gilson Chromatography system using 0.1% AcOH / CH ₃ CN / H ₂ O.

Example FF)

The product of Example FF-5 (100 mg, 0.0005 mol) was dissolved in 8N HCl (20 mL) and stirred under reflux for 10 hours. The reaction mixture was cooled to room temperature and the aqueous HCl was evaporated in a rotary evaporator. The residue was dissolved in deionized water (10 mL) and water and re-concentrated in vacuo to give the title product as a yellow glassy solid in near quantitative yield (88 mg).

Example GG

(2R / S, 4Z) -2-Amino-2-methyl-7-[(1-iminoethyl) amino] -4-heptenoic acid, dihydrochloride

Example GG-1)

5,6 dihydropyran-2-one (49.05 g, 0.5 mole) was dissolved in 200 mL of water. Potassium hydroxide (35 g, 0.625 mol) was added and the reaction mixture was stirred for 5 hours under ambient temperature. The solvent was removed in vacuo to give a colorless glassy solid (65 g, 84%), which was found to be mainly the cis isomer of the title compound by NMR.

Example GG-2)

The product of Example GG-1 was dissolved in 100 ml of dimethyl formamide. Then, methyl iodide (52 mL, 0.84 mole) was added, resulting in an exothermic reaction at 40 ° C. The reaction mixture was stirred for 10 hours at room temperature and partitioned between 150 mL of 20/80 ratio ethylacetate / diethylether and ice water. The aqueous layer was separated and extracted again with 100 mL diethyl ether. The organic layers were combined, dried (Na ₂ S0 ₄ ), filtered and all solvents stripped to afford the desired methyl ester product (40 g, 71%). This material was dissolved in 200 ml of methylene chloride and the solution was cooled to 0 ° C. t-butyldimethylsilylchloride, triethylamine and dimethylaminopyridine were added. The reaction mixture was slowly warmed to room temperature and stirred under nitrogen for 10 hours. The reaction was extracted with 100 mL of 1N aqueous potassium disulfide solution. The organic layer was washed with brine (2 x 100 mL) and then with water (3 x 150 mL). The organic layer was dried (Na ₂ SO ₄ ), filtered and stripped to give 42 g (56%) of the title material.

Example GG-3)

The material from Example GG-2 was dissolved in 25 ml of toluene and cooled to 0 ° C. Diisobutylaluminum hydride (1.0 M in toluene, 32 mL, 48 mmol) was added dropwise while maintaining the temperature between 5 and -10 ° C. The reaction mixture was stirred for 1.5 h at a temperature of 6-8 ° C. and then cooled to −25 ° C. To this mixture was added 100 mL of 0.5 N sodium potassium tartarate. The reaction mixture was allowed to warm to rt and stirred for 1 h. A gelatinous precipitate formed which was filtered off. The aqueous material was extracted with EtOAc (2 x 100 mL). The combined organic layers were dried (sodium sulfate), filtered and concentrated in vacuo to afford the title product as a colorless oil (3.45 g, 66%).

Example GG-4)

The product from Example GG-3 (8 g, 37 mmol) was dissolved in 100 ml of methylene chloride and the solution was cooled to 0 ° C. Then methanesulfonyl chloride was added and the mixture was stirred for 5 minutes. Then triethylamine was added. During the addition of the reagents described above, the temperature was maintained between 0 and -10 ° C. The reaction mixture was subsequently warmed to room temperature and stirred for 24 hours. Then, extracted with 100 ml of 50% aqueous sodium bicarbonate solution. The organic layer was washed with 100 mL of saturated brine aqueous solution, dried (sodium sulfate), filtered and stripped in vacuo to afford the title material (8.2 g, 94%).

Example GG-5)

A solution of N-p-chloro phenylimine alanine methyl ester (8.85 g, 34 mmol) dissolved in 59 mL tetrahydrofuran was purged with argon. NaH (1.64 g, 41 mmol) was added and the solution turned bright orange and then dark red. A solution of the title material (8 g, 34 mmol) of Example GG-4 in 40 mL tetrahydrofuran was added to the anionic solution. An exothermic reaction was observed indicating a temperature rise to nearly 40 ° C. The reaction mixture was maintained at 48 to -52 ° C for 2 hours. Then cooled to room temperature and filtered. The filtrate was stripped under vacuum to afford the title material (8.4 g, 50% crude yield) as a yellow oil.

Example GG-6)

The title material (8.4 g, 18.2 mmol) of Example GG-5 was treated with 125 mL of 1N hydrochloric acid and the reaction was stirred for 1 h at room temperature. After ethyl acetate was extracted from the reaction mixture (2 × 75 mL), the aqueous layer was stripped under vacuum at 56 ° C. to give 4 g of the title material (100% crude yield).

Example GG-7)

The title product (1.9 g, 8.5 mmol) of Example GG-6 was dissolved in a mixture of 15 mL of dioxane and 8 mL of water. Then, the solid potassium bicarbonate was carefully added to prevent foaming. After the reaction mixture was stirred for 10 minutes, t-butyloxycarbonyl anhydride was added dropwise and the reaction mixture was stirred for 24 hours under ambient temperature. The reaction mixture was diluted with 100 ml of ethyl acetate and 50 ml of water, which was then poured into a separating funnel. The organic layer was separated, dried (Na ₂ SO ₄ ), filtered and stripped to give the title material as a colorless oil (1.9 g, 78% crude yield).

Example GG-8)

Another 1.9 g sample of the title material obtained from Example GG-6 was converted to a crude Z / E mixture of the title product of Example GG-7 by the method of Example GG-7. This material was further purified on silica using a solvent system of 20/80 ethylacetate / hexanes to give small amounts of E-isomers and most of the Z-isomers.

Example GG-9)

The title Z-isomer (1.8 g, 6.25 mmol) from Example GG-8 was dissolved in 20 ml of acetonitrile and the solution was cooled to 0 ° C. Then pyridine (0.76 g, 9.4 mmol) was added followed by dropwise addition of solid dibromotriphenylphosphorane (3.46 g, 8.2 mmol) over 10 minutes. The reaction mixture was stirred under argon for 24 hours under room temperature. The precipitate formed was filtered off. The filtrate was concentrated in vacuo to afford 2.8 g of oil, which was purified on silica gel using a solvent system of 60/40 ethyl acetate / hexanes. 1.1 g of the title material (50%) was characterized by NMR.

Example GG-10)

The title material (300 mg, 0.86 mmol) from Example GG-8 was dissolved in 25 mL dimethylformamide (DMF). Potassium salt of 3-methyl-1,2,4-oxadiazolin-5-one (130 mg, 0.94 mmol) was added and the reaction mixture was heated to 52 ° C. and maintained for 18 hours with stirring. Then, after cooling to room temperature, the DMF was stripped at 60 ° C. under vacuum. The residue was purified on silica gel using 60/40 to 90/10 gradient ethyl acetate / hexanes to give 300 mg (95%) of the title material.

Example GG-11)

The product of Example GG-10 (300 mg) was treated with 0.05 N aqueous HCl and the solution was stirred for 30 minutes. The solvent was removed in vacuo to give the desired material in near quantitative yield.

Example GG-12)

The title material (198 mg, 0.54 mmol) from Example GG-11 was dissolved in 50 mL MeOH. Formic acid (40 mg) was then added followed by palladium on calcium carbonate (400 mg). The reaction mixture was heated to 65 ° C. with stirring in a sealed tube for 24 hours. Then cooled to room temperature and filtered. The filtrate was concentrated in vacuo and the residue was purified by reverse phase HPLC to yield 115 mg (75%) of the title material.

Example GG)

The title material (75 mg) from Example GG-12 was dissolved in 15 mL 2N hydrochloric acid. The reaction mixture was heated to reflux, stirred for 6 hours and then cooled to room temperature. Solvent was removed in vacuo. The residue was dissolved in 25 ml of water and stripped on a rotary evaporator to remove excess hydrochloric acid. The residue was dissolved in water and lyophilized to yield 76 mg (about 100%) of the title material.

Example HH

(2S, 5E) -2-Amino-2-methyl-6-fluoro-7-[(1-iminoethyl) amino] -5-heptenoic acid, dihydrochloride

Example-HH-1)

To a low temperature (-78 ° C.) solution of triethyl 2-fluorophosphonoacetate (25.4 g, 105 mmol) in 100 mL of THF was added n-butyl lithium (63 mL, 1.6 M in hexane, 101 mmol). The mixture was stirred for 20 minutes at -78 ° C, resulting in a light yellow solution. Then a solution of 120 mL THF crude 3-[(t-butyldimethylsilyl) oxy] propanal ("J. Org. Chem., 1994, 59, 1139-1148") (20.0 g, 105 mmol) ) Was added dropwise over 10 minutes and the resulting mixture was stirred at −78 ° C. for 1.5 hours, at which time analysis by thin layer chromatography (5% ethyl acetate in hexane) showed no starting material remaining. The reaction was quenched with saturated aqueous NH ₄ Cl (150 mL) at −78 ° C. The organic layer was collected and the aqueous layer was extracted with diethyl ether (300 mL). The combined organics were washed with brine (200 mL), dried over MgSO ₄ , filtered and concentrated. The crude material was filtered through a plug of silica gel (150 g) eluting with hexane (2 L) to give the desired (2E) -5-[[(1,1-dimethylethyl) dimethylsilyl] oxy] -2. 14.38 g (52%) of -fluoro-2-pentenoic acid ethyl ester product was obtained as a clear oil. ¹ H NMR and ¹⁹ F NMR showed that the isolated product had an E: Z ratio of about 95: 5.

Example-HH-2)

To a solution of Example-HH-1 (6.76 g, 24.5 mmol) in 100 mL methanol was added 1.4 g of solid NaBH ₄ (4.2 g, 220 mmol) over 3 hours at room temperature. After 3.5 hours, water was added (10 mL). Additional solid NaBH ₄ (4.2 g, 220 mmol) was added in 1.4 g portions over 3 hours. The reaction was quenched with 150 mL saturated aqueous NH ₄ Cl and extracted with diethyl ether (2 × 250 mL). The organic layers were combined, dried over MgSO ₄ , filtered and concentrated. 4.81 g of the crude material, clear oil, was purified by flash column chromatography on silica gel eluting with 10% ethyl acetate in hexanes to give the desired (2E) -5-[[(1,1-dimethylethyl) di 2.39 g (42%) of methylsilyl] oxy] -2-fluoro-2-penten-l-ol product was obtained as a clear oil, which was found to have an E: Z ratio of 93: 7 by ¹⁹ F NMR. .

Example-HH-3)

Example-HH-2 (2.25 g, 9.58 mmol), polymer-supported triphenylphosphine (3 mmol / g, 1.86 g, 15 mmol) and 3-methyl-1,2,4oxadia in 60 mL THF To a mixture of zolin-5-one (1.25 g, 12.5 mmol) diethylazodicarboxylate (2.35 mL, 14.7 mmol) was added dropwise. The reaction mixture was stirred under rt for 1 h and additional 3-methyl-1,2,4-oxadiazolin-5-one (0.30 g, 3.0 mmol) was added. After 30 minutes, the mixture was filtered through celite and the filtrate was concentrated. The resulting yellow oil was triturated with diethyl ether (30 mL) and the solids were removed by filtration. The filtrate was concentrated, triturated with hexane (30 mL) and filtered. The filtrate was concentrated to give an oil which was purified by flash column chromatography on silica gel eluting with 15% ethyl acetate in hexanes to give the desired 4-[(2E) -5-[[(1,1-dimethyl Ethyl) dimethylsilyl] oxy] -2-fluoro-2-pentenyl] -3-methyl-1,2,4-oxadiazol-5 (4H) -one product 1.83 g (60%) clear oil ¹⁹ F NMR showed that it only contained the desired E-isomer.

Example-HH-4)

A solution of Example-HH-3 (1.83 g, 5.78 mmol) in a mixture of acetic acid (6 mL), THF (2 mL) and water (2 mL) was stirred for 2.5 h at room temperature. The resulting solution was concentrated in vacuo to afford an oil, which was dissolved in diethyl ether (50 mL). The organic layer was washed with saturated NaHCO ₃ , the aqueous layer was extracted with diethyl ether (2 × 50 mL) and extracted with ethyl acetate (2 × 50 mL). The combined organic layers are dried (MgSO ₄ ), filtered and evaporated to the desired 4-[(2E) -2-fluoro-5-hydroxy-2-pentenyl] -3-methyl-1,2,4 1.15 g (98%) of oxadiazole-5 (4H) -one product was obtained as a clear colorless oil.

Example-HH-5)

To a CH ₂ Cl ₂ (2 mL) solution of triphenylphosphine (238 mg, 0.91 mmol) and imidazole (92 mg) was added solid iodine (230 mg, 0.91 mmol) at 0 ° C. and the mixture was stirred for 5 minutes. Stirred. To the resulting yellow slurry was added a solution of Example-HH-4 (0.15 g, 0.74 mmol) CH ₂ Cl ₂ (1.5 mL). The slurry was warmed to room temperature and stirred for 30 minutes. The reaction mixture is diluted with CH ₂ Cl ₂ (10 mL), washed with saturated Na ₂ S ₂ O ₃ (5 mL) and brine (5 mL), dried (MgSO ₄ ), filtered and evaporated to give an oil. Obtained. Diethyl ether (10 mL) was added to the oil to give a white precipitate, which was removed by filtration and the filtrate was concentrated to give an oil. The crude material is purified by flash column chromatography on silica gel eluting with 30% ethyl acetate in hexane to give the desired 4-[(2E) -2-fluoro-5-iodo-2-pentenyl] -3 0.18 g (78%) of -methyl-1,2,4-oxadiazol-5 (4H) -one product was obtained as a clear oil, which solidified on standing. Melting point = 58.1 to 58.6 ° C.

Example-HH-6)

(3S, 6R) -6-isopropyl-3-methyl-5-phenyl-3,6-dihydro-2H-1,4-oxazin-2-one in an ice bath (Synthesis, 1999, 4, 704-717 ") (1.10 g, 4.76 mmol), Li-I (0.63 g, 4.76 mmol) and Example-HH-5 (0.85 g, 2.72 mmol) solution of 1-methyl-2-pyrrolidinone (12 mL) To 2-t-butylimino-2-diethylamino-1,3-dimethylperhydro-1,3,2-diazaphosphorine (1.38 mL, 4.76 mmol) was added. The yellow solution turned orange upon addition of base and the resulting solution was stirred for 1 h under room temperature. The reaction mixture was diluted with ethyl acetate (100 mL), washed with water (2 x 30 mL), dried (MgSO ₄ ), filtered and evaporated to give a yellow oil. The crude material was purified by flash column chromatography on silica gel eluting with 30% ethyl acetate in hexanes to give 0.64 g (57%) of the desired alkylation product as a clear oil.

Example-HH-7)

Example-Lindla catalyst (1.0 g) was added to a methanol (20 mL) solution of HH-6 (0.13 g, 0.31 mmol). The stirred slurry was heated to 60 ° C. for 1 hour and additional Lindla catalyst (0.30 g) was added. The slurry was further stirred at 60 ° C. for 1 hour and then cooled to room temperature. The catalyst was removed by filtration through celite and the filtrate was stripped to yield 0.58 g (100%) of the desired deprotected amidine product as pale yellow oil.

Example-HH)

A solution of the product of Example-HH-7 (0.58 g, 1.54 mmol) in 1.5 N HCl (25 mL) was washed with diethyl ether (2 x 20 mL) and refluxed for 1 h. The solvent was stripped off and the crude amino acid ester was dissolved in 6N HCl (15 mL) and heated to reflux. After 6 hours, the solvent was removed in vacuo and the resulting foam was purified by reverse phase HPLC eluting with a 30 minute gradient of 0-40% CH ₃ CN / H ₂ O (0.25% acetic acid). Fractions containing product were combined and concentrated to give a foam. The product was dissolved in 1N HCl and the solvent removed in vacuo (2 ×) to give the desired (2S, 5E) -2-amino-2-methyl-6-fluoro-7-[(1-iminoethyl) amino] 0.15 g (29%) of -5-heptenoic acid, dihydrochloride product were obtained.

Example II

(2S, 5E) -2-Amino-2-methyl-6-fluoro-7-[(1-iminoethyl) amino] -5-heptenoic acid, dihydrochloride

Example-II-1)

To a solution of methyl N-[(3,4-dichlorophenyl) -methylene] -alanine (748.5 g, 2.88 moles) of 1-methyl-2-pyrrolidinone (7500 mL) under LiI (385.5 g, 2.88 mole) was added and the resulting slurry was stirred for about 20 minutes to yield a clear solution. Then, solid (750 g, 2.40 mole) from Example-HH-5 was added and the resulting solution was cooled to about 0 ° C. in an ice bath. Pure BTPP (900 g, 2.88 moles) was added dropwise over 25 minutes while maintaining the internal temperature below 5 ° C. After 1.5 hours of further stirring at 5 ° C., it was determined by HPLC whether the reaction was complete. At this point, 7500 ml of methyl t-butyl ether (MTBE) was added, followed by 9750 ml of a mixture of water / crushed ice. The temperature was raised to 20 ° C. during this working period. After vigorous stirring for 5 to 10 minutes, the layers were separated and the aqueous layer was washed twice with 6000 mL MTBE. The MTBE layers were combined and washed twice with 7500 mL of water. The resulting MTBE solution was then concentrated to about 5000 ml, treated with 11625 ml of 1.0 NHCl and stirred vigorously under room temperature for 1 hour. The layers were separated and the aqueous layer was washed with 7500 mL MTBE. About 1 kg of sodium chloride was added to the aqueous layer and the resulting mixture was stirred until all salts dissolved. At this point, 7500 mL of ethyl acetate was added and the resulting mixture was cooled to 10 ° C. and 2025 mL of 6.0 N sodium hydroxide was added with good shaking. The resulting pH should be about 9. The layers were separated and the aqueous layer was saturated with sodium chloride and extracted again with 7500 mL ethyl acetate. The combined ethyl acetate extracts were dried (MgSO ₄ ) and concentrated to give as a bright oil. It should be noted that the ethyl acetate was not completely removed. Then, 3000 ml of hexane was added to give a slurry, which was cooled to 10 ° C. The granular solid was collected by filtration and washed with 1500 ml of hexane. About 564 g (82% yield) of the desired pure aminoester (> 95% purity by HPLC) was obtained as a white solid. Melting point 82.9 to 83.0 ° C. LCMS: m / z = 288.2 [M + H] ⁺ . Chiral HPLC (Chiralpak-AD normal phase column, 100% acetonitrile, 210 nm, 1 ml / min): major peak is present at 4.71 and 5.36 min (1: 1).

Example-II-2)

Separate enantiomers of the product from Example-II-1 on a production scale using chiral HPLC chromatography (ChiralPak-AD, top column, 100% acetonitrile) to separate the desired pure (2S) -2-methyl Amino ester product The title product was obtained. ChiralPak-AD, top column, 100% acetonitrile, 210 nm, 1 mL / min): 5.14 min (99%).

Example-II-3)

A slurry of the product of Example-II-2 (2.30 g, 8.01 mmol) in 0.993 M NaOH (30.0 mL, 29.79 mmol) was stirred for 2 h at room temperature. To the resulting clear colorless solution was added 1.023 M HCl (29.10 mL, 29.76 mmol). The resulting clear solution was concentrated until a precipitate began to form (about 30 mL). The slurry was warmed to give a clear solution which was left overnight at room temperature. The precipitate was isolated by filtration. The solid was washed with cold water (2 × 10 mL), cold methanol (2 × 10 mL) and Et ₂ O (2 × 20 mL). The white solid was dried under vacuum at 40 ° C. for 4 hours to yield 1.04 g (53%) of the desired N-hydroxy ashed product. Melting point = 247.2 ° C.

Example-II-4)

To the solution of Example-II-3 in methanol is added a Lindla catalyst. The stirred slurry is refluxed for 2 hours and then cooled to room temperature. The catalyst is removed by filtration through celite and the filtrate is stripped. The resulting solid was dissolved in water and concentrated repeatedly from 1.0 N HCl to give the desired (2R, 5E) -2-amino-2-methyl-6-fluoro-7-[(1-iminoethyl) amino] -5-heptenoic acid, dihydrochloride product is obtained.

Example-II-5)

A solution of 73.5 g (0.3 mole) of the product from Example-II-2 was dissolved in 300 mL of methanol and 13.7 g of Lindla catalyst and 3 formic acid (1.53 mole) in 312 mL of methanol while maintaining the reaction temperature at 22-26 ° C. 73.5 g of the preformed mixture was added dropwise. After about 15 hours more stirring at room temperature, it was determined by ¹⁹ F NMR whether the reaction was complete. The resulting reaction mixture was filtered through celite and the celite was washed three times with 125 ml of methanol. The methanol filtrates were combined and concentrated to give 115 g of the desired amidine title product as a viscous oil.

To remove small amounts of lead, the crude product was dissolved in 750 ml of methanol and 150 g of thiol-based resin (Deloxan THP 11) was added. After stirring for 3 hours at room temperature, the resin was filtered and washed twice with 500 ml of methanol. The filtrate was collected and concentrated to give 99 g of the desired amidine title product as a viscous oil.

Another example:

A total of 5.0 g (0.0174 mol, 1.0 equiv) of product from Example-II-2 were mixed with 40 g of 1-butanol and 5.0 g of zinc powder (0.0765 mol, 4.39 equiv) in 10 mL of acetic acid. After stirring at 50 ° C. for 5 hours, LC analysis showed the reaction to be complete. The solid was easily filtered. After cooling to 7 ° C. in ice water, the filtrate was treated once with 30 mL of 6N NaOH (0.180 mole) with vigorous stirring. After the reaction mixture was cooled to 33-20 [deg.] C., the clear butanol layer was separated off and the aqueous layer was extracted again with 40 mL of 1-butanol. Butanol extracts were combined, washed with 30 mL of brine and then with about 10 mL of 6N HCl. After concentration at 70 ° C., a clear glass was produced, which turned out to be the desired amidine title product.

Example-II)

A solution of 99 g of the product from Example-II-5 in 6N HCl was refluxed for 1 hour at which time LC analysis showed the reaction to be complete. The solvent was removed in vacuo to yield 89.2 g of glassy oil, which was dissolved in a mixture of 1466 mL ethanol and 7.5 mL deionized water. THF was added to the stirred solution under ambient temperature (5.5 L) until the cloud point was reached. More 30 ml of deionized water was added and the solution was shaken overnight at room temperature. The resulting slurry was filtered and washed with 200 mL of THF to give 65 g of a white solid which proved to be the desired title product.

Example JJ

(2R, 5E) -2-Amino-2-methyl-6-fluoro-7-[(1-iminoethyl) amino] -5-heptenoic acid, dihydrochloride

Example-JJ-1)

Chiral HPLC chromatography was used to separate the individual enantiomers of the product from Example-II-1 on a production scale to afford the desired pure (2R) -2-methyl amino ester product.

Example-JJ-2)

The product from Example-JJ-1 was dissolved in water and acetic acid. Zinc powder was added and the mixture was heated to 60 ° C. until HPLC analysis indicated that little starting material remained. Zn was filtered through celite from the reaction mixture and the filtrate was concentrated. The crude material was purified by reverse phase HPLC column chromatography. Fractions containing product were combined and concentrated to afford the desired (2R) -2-methyl acetamidine product.

Example-JJ)

The solution of Example-JJ-2 in 2.0 N HCl is refluxed for 2 hours. Solvent was removed in vacuo. The resulting solid was dissolved in water and concentrated repeatedly from 1.0 N HCl to give the desired (2R, 5E) -2-amino-2-methyl-6-fluoro-7-[(1-iminoethyl) amino] -5-heptenoic acid, dihydrochloride product was obtained.

Example KK

(2R / S, 5E) -2-Amino-2-methyl-6-fluoro-7-[(1-iminoethyl) amino] -5-heptenoic acid, dihydrochloride

Example-KK-1)

Samples of the product of methyl N-[(4-chlorophenyl) methylene] -glycinate (0.33 g, 1.6 mmol), LiI (0.20 g, 1.0 mmol) and Example-HH-5 (0.30 g, 0.96 mmol) in 1-methyl-2-pyrrolidinone (5 mL) solution, 2-t-butylimino-2-diethylamino-1,3-dimethylperhydro-1,3,2-dia Zaposporin (0.433 mL, 1.5 mmol) was added. The solution was stirred for 1.5 h at room temperature. The reaction mixture is diluted with ethyl acetate (30 mL), washed with water (2 x 20 mL), dried (MgSO ₄ ), filtered and evaporated to afford the crude desired racemic alkylated imine as a yellow oil. It was.

The crude material was dissolved in ethyl acetate (10 mL) and 1N HCl (10 mL) was added. The mixture was stirred for 2 h under room temperature and the organic layer was separated. The aqueous layer was neutralized with solid NaHCO ₃ and extracted with ethyl acetate (2 × 30 mL). The organic layer was dried (MgS0 ₄ ), filtered and evaporated to afford 0.13 g of the desired title racemic amino ester product as a yellow oil. This product was used without further purification in the next step.

LCMS: m / z = 288.2 [M + H] ⁺ .

Example-KK-2)

Example- To a solution of CH ₂ Cl ₂ (15 mL) of KK-1 (1.36 g, 4.98 mmol), 4-chlorobenzaldehyde (0.70 g, 5.0 mmol) and MgSO ₄ (about 5 g) were added. The slurry was stirred for 18 hours under room temperature. The slurry was filtered and the filtrate was stripped to yield 1.98 g (100%) of the desired title imine product as a pale yellow oil. This product was used without further purification in the next step.

Example-KK-3)

Example-Methyl iodide (0.200 mL, 3.23 mmol) and O (9) -allyl-N- (9-) in a solution of CH ₂ Cl ₂ (2 mL) of the product of KK-2 (0.25 g, 0.63 mmol) Anthracenylmethyl) -cinconidinium bromide (40 mg, 0.066 mmol) was added. The solution was cooled to -78 ° C and pure BTPP (0.289 mL, 0.95 mmol) was added. The resulting orange solution was stirred at -78 ° C for 2 hours and brought to -50 ° C. After being at −50 ° C. for 2 hours, the solution is diluted with CH ₂ Cl ₂ (10 mL), washed with water (10 mL), dried (MgSO ₄ ), filtered and evaporated to give the crude desired racemic Alkylated imine was obtained as a yellow oil.

The crude material was dissolved in ethyl acetate (10 mL) and 1N HCl (10 mL) was added. The mixture was stirred for 1 h under room temperature and the organic layer was separated. The aqueous layer was neutralized with solid NaHCO ₃ and extracted with ethyl acetate (2 × 30 mL). The organic layer was dried (MgSO ₄ ), filtered and evaporated to give 0.16 g of the desired racemic 2-methylamino ester product. Obtained as a yellow oil. The product was used without further purification in the next step. LCMS: m / z = 288.2 [M + H] ⁺ .

Example-KK-4)

The racemic product from Example-KK-3 is dissolved in water and acetic acid. Zinc powder is added and the mixture is heated to 60 ° C. until HPLC analysis shows that little starting material remains. Zn powder is filtered from the reaction mixture through celite and the filtrate is concentrated. The crude material is purified by reverse phase HPLC column chromatography. The fractions containing the product are combined and concentrated to give the desired acetamidine product.

Example-KK)

The solution of racemate Example-KK-4 in 2.0 N HCl is refluxed for 1 hour. The solvent is removed in vacuo. The resulting solid was dissolved in water and concentrated repeatedly from 1.0 N HCl to give the desired title (2R / S, 5E) -2-amino-2-methyl-6-fluoro-7-[(1-iminoethyl ) Amino] -5-heptenoic acid, dihydrochloride product.

Example LL

(2S, 5Z) -2-Amino-2-methyl-7-[(1-iminoethyl) amino] -5-heptenoic acid, dihydrochloride

4-[(tetrahydropyranyl) oxy] butene

Example LL-1)

A mixture of 4-dihydro-2H-pyridine (293.2 g 3.5 mol) and concentrated HCl (1.1 mL) was cooled to 5 ° C. While continuing to cool externally, 3-butyn-1-ol (231.5 g, 3.3 mol) was added over 30 minutes and the temperature reached 50 ° C. The reaction was kept mixing for 2.5 hours at room temperature and then diluted with MTBE (1.0 L). The resulting mixture was washed with saturated sodium bicarbonate (2 x 150 mL). The organic phase was dried over sodium sulfate and concentrated under reduced pressure to yield 500 g (98% crude yield) of product; GC area%: 96%.

5- (tetrahydro-pyran-2-yloxy) -pent-2-yn-1-ol

Example LL-2)

To a solution of the 4-[(tetrahydropyranyl) oxy] butene product of Example LL-1 (50.0 g, 0.33 mol) in THF (125 mL), a solution of 2N EtMgCl in THF (242 mL, 0.48 mol) was added. It was added over 30 minutes under a nitrogen atmosphere, which raised the temperature to 48 ° C. The mixture was further heated to 66 ° C. and maintained at this temperature for 2 hours before cooling to ambient temperature. Paraformaldehyde (14.5 g, 0.48 mol) was added (small exothermic reaction was observed) and the resulting mixture was heated to 45 ° C. After adjusting the temperature to 45-55 [deg.] C. for 1 hour, the mixture turned clear. At this point, the mixture was heated to 66 ° C. and stirred for 2.5 h. The mixture was cooled to room temperature, saturated ammonium chloride (125 mL) was added slowly over 30 minutes (large exotherm was observed) and the temperature was kept below 40 ° C. The liquid phase was discarded by separation and ethyl acetate (250 mL) and brine (50 mL) were added. The organic phase was separated and washed with brine (2 × 50 mL) and water (1 × 50 mL). The organic layer was dried over sodium sulfate and concentrated under reduced pressure to give 51 g as a light yellow oil (85% crude yield); GC area% = 88% title product, 6% starting material.

5- (tetrahydro-pyran-2-yloxy) -pent-2-en-1-ol

Example LL-3)

5- (tetrahydro-pyran-2-yloxy) -pent-2-yn-1-ol product of Example LL-2 (40.2 g, 0.22 mol) in a 500 ml Parr bottle under nitrogen atmosphere, Lindla catalyst (2.0 g), ethanol (120 mL), hexane (120 mL) and 2,6 lutidine (457 mg) were charged. The reaction mixture was purged five times with nitrogen and hydrogen gas. The Par bottle was pressurized to 5 psi with hydrogen and shaken until 98% of theoretical hydrogen was consumed. Hydrogen was released from the vessel and the reaction was purged five times with nitrogen. The mixture was filtered through a pad of Solka Floc and the catalyst was rinsed with ethanol (2 x 50 mL). The filtrate and rinse were combined and concentrated under reduced pressure to give 40.3 g (99% yield) of the title material as a yellow oil (GC area% = 96%).

3-methyl-4- [5- (tetrahydro-pyran-2-yloxy) -pent-2-enyl] -4H- [1,2,4] oxadiazol-5-one

Example LL-4)

To a solution of the 5- (tetrahydro-pyran-2-yloxy) -pent-2-en-1-ol product of Example LL-3 (11.8 g, 0.063 mol) in toluene (42 mL) was treated with triethylamine ( 6.4 g, 0.063 mol) was added. The mixture was cooled to -5 ° C and methanesulfonyl chloride (7.3 g, 0.63 mole) was added via syringe at a rate to keep the pot temperature below 10 ° C. The mixture was allowed to warm to rt and stirred for 2 h. The mixture was filtered by suction and rinsed with toluene on the filter (2 × 20 mL). The filtrate and washings were added to a mixture of sodium salts of 3-methyl-1,2,4-oxadiazolin-5-one (8.6 g, 0.063 mol) in DMF (10 mL). The mixture was stirred with a mechanical stirrer and heated at 45 ° C. for 5 hours. Water (40 mL) was added and the mixture was stirred for 5 minutes, then the layers were separated. The toluene layer was washed with water (3 × 20 mL), dried over MgSO ₄ and concentrated to give 16.5 g (97.3%) of orange crude product (area% GC was 71% title product, 18% toluene and 4% impurities). Consists of).

4- (5-hydroxy-pent-2-enyl) -3-methyl-4H- [1,2,4] oxadiazol-5-one

Example LL-5)

3-Methyl-4- [5- (tetrahydro-pyran-2-yloxy) -pent-2-enyl] -4H- [1 of Example LL-4 (16 g, 0.06 mol) in methanol (48 mL) To the solution of the, 2,4] oxadiazol-5-one product was added p-toluenesulfonic acid (0.34 g, 2.0 mmol). The mixture was stirred for 4 hours under room temperature. Sodium bicarbonate (0.27 g, 3.0 mmol) was added and the mixture was evaporated on a rotary evaporator. The residue was diluted with saturated NaHCO ₃ (20 mL) and the resulting mixture was extracted with ethyl acetate (2 × 60 mL). The extracts were combined, washed with water (2 × 25 mL), dried over MgSO ₄ and concentrated to yield 8.4 g of crude orange oil title product (area% GC = 80%).

Methanesulfonic acid 5- (3-methyl-5-oxo- [1,2,4] oxadiazol-4-yl) -pent-3-enyl ester

Example LL-6)

4- (5-hydroxy-pent-2-enyl) -3-methyl-4H- [1,2,4] oxadia of Example LL-5 (8.27 g, 0.045 mol) in methylene chloride (33 mL) To the solution of sol-5-one product was added triethylamine (5.0 g, 0.49 mol). The mixture was cooled to -5 ° C and methanesulfonyl chloride (5.5 g, 0.048 mole) was added to maintain the temperature below 8 ° C. The cold bath was removed and the mixture was stirred for 3 hours while warming to room temperature. Water (15 mL) was added and the mixture was stirred for 5 minutes, then the layers were separated. The organic phase was washed with water (10 mL), dried over MgSO ₄ and concentrated to give a light amber residue. The residue was dissolved in ethyl acetate (8 mL) and kept at 5 ° C. overnight. The precipitated solid was filtered off with suction, rinsed with a minimum amount of ethyl acetate on the filter and then air dried on the filter to give 6.8 g (58% yield) of the title product.

4- (5-iodo-pent-2-enyl) -3-methyl-4H- [1,2,4] oxadiazol-5-one

Example LL-7)

Methanesulfonic acid 5- (3-methyl-5-oxo- [1,2,4] oxadiazol-4-yl) -pent of Example LL-6 (20.0 g, 0.076 mol) in acetone (160 mL) To the solution of the 3-enyl ester product was added sodium iodide (17.15 g, 0.114 mol). The mixture was heated to reflux and stirred for 3 hours. External heating was stopped and the mixture was kept overnight at room temperature. The solid was removed by filtration and rinsed on the filter. The filtrate and washes were combined, concentrated and the heterogeneous residue was extracted with ethyl acetate (120 mL). The organic layer was washed with water (60 mL) and washed with 15% aqueous solution of sodium thiosulfate (60 mL) and water (60 mL); Dry over MgSO ₄ and concentrate under reduced pressure to give 22.1 g (98% yield) of the title oil product.

2-[(3,4-Dichloro-benzylidene) -amino] -propionic acid methyl ester

Example LL-8)

To a mechanically stirred slurry (200.0 g, 1.43 mol) of L-alanine methyl ester hydrochloride in methylene chloride (2.1 L) was added triethylamine (199.7 ml, 1.43 mol) over 12 minutes under nitrogen atmosphere (additional After some dissolution of the solids, during reprecipitation). After 10 minutes, 3,4-dichlorobenzaldehyde (227.5 g, 1.30 moles) and magnesium sulfate (173.0 g, 1.43 moles) were added (temperature rose 6 ° C. over 30 minutes). After 2.5 hours, the mixture was filtered. The filtrate was washed with water (1 × 1 L) and brine (1 × 500 mL), dried over sodium sulphate, filtered and concentrated to give an oil product in 313.3 g, 92.4% yield.

Rac-2-amino-2-methyl-7- (3-methyl-5-oxo- [1,2,4] oxadiazol-4-yl) -hept-5-enoic acid methyl ester

Example LL-9)

Method 1.

A solution of the product of Example LL-7 (114.2 g, 0.39 mol) and the product of Example LL-8 (151.5 g, 0.58 mol) in dimethylformamide (1.4 L) was cooled to -8 ° C under nitrogen atmosphere. . Lithium iodide (78.1 g, 0.58 mol) was then added in equal portions three times and added over 19 minutes. The mixture was stirred at −7 ° C. for 20 minutes, then (t-butylimino) -tris (pyrrolidino) phosphorane (194.0 mL, 0.62) was added over 36 minutes (maximum temperature = −2.6 ° C.) ). After 10 minutes, the cooling bath was removed and the solution was stirred for 1 hour under ambient temperature. The mixture was then poured into cold water (1.4 L) and extracted with ethyl acetate (2 x 1.0 L). The combined organic layers were washed with water (2 x 400 mL) and brine. The ethyl acetate layer was treated with 1N HCl (780 mL) and stirred for 1 hour. The aqueous layer was separated and extracted with ethyl acetate (2 x 400 mL) and then neutralized with sodium bicarbonate (110 g). The mixture was extracted with ethyl acetate (1 x 500 mL). The organic layer was dried over sodium sulphate, filtered, concentrated and treated with methyl t-butyl ether to give crystalline product: first yield 14.4 g; Second yield 6.6 g (GC purity = 96.2 and 91.9%, respectively). The aqueous phase was saturated with sodium chloride and extracted with ethyl acetate (4 x 500 mL). The combined organic layers were dried over sodium sulphate, filtered, concentrated and treated with methyl t-butyl ether to give crystalline product: first yield 33.4 g; Second yield 10.8 g (GC purity = 89.6 and 88.8% respectively. Total crude yield 65.2 g, 62.4%).

Method 2.

In a solution of the product of Example LL-7 (20.7 g, 0.070 mol) and the product of Example LL-8 (22.9 g, 0.088 mol) in dimethylformamide (207 mL) cesium carbonate (29.8 g, 0.092) was added. The mixture was stirred at rt for 16 h, then diluted with water (300 mL) and extracted with ethyl acetate (2 x 200 mL). The combined ethyl acetate layers were washed with water (3 x 100 mL) and brine and then treated with 1N HCl (184 mL). After 1 hour, the layers were separated and the aqueous layer was extracted with ethyl acetate (3 × 100 mL) and then neutralized with sodium bicarbonate (15.5 g). The mixture was extracted with ethyl acetate (1 x 150 mL). The aqueous layer was saturated with sodium chloride and extracted with ethyl acetate (3 x 100 mL). The combined organic layers were dried over sodium sulphate, filtered and concentrated to give 11.9 g, 62.9% of a yellow solid; GC purity = 96.6%. The crude product was recrystallized from warm methyl t-butyl ether or ethyl acetate.

Rac-2-Amino-2-methyl-7- (3-methyl-5-oxo- [1,2,4] oxadiazol-4-yl) -hept-5-enoic acid

Example LL-10)

The product of Example LL-9 (0.269 g, 1 mmol) was dissolved in 5 mL 2N HCl and heated to reflux under argon. After refluxing for 6 hours and stirring for 72 hours at room temperature, an aliquot was removed and checked by ¹ H NMR. About 6% of unreacted starting ester remained with the desired product (validated by LC-MS). The aqueous portion was removed in vacuo to yield 0.38 g as a dark amber oil. After purification by reverse phase chromatography and lyophilization, 0.23 g, 90.2% of the title compound were obtained as a white, non-deliquescent solid.

(2S, 5Z) -2-Amino-2-methyl-7- (3-methyl-5-oxo- [1,2,4] oxadiazol-4-yl) -hept-5-enoic acid methyl ester

Example LL-11)

The title compound (827.3 g) was separated from its R enantiomer by preparative chiral chromatography using Novaprep 200 instrument with stationary recycling options. The material was dissolved in anhydrous ethanol at a concentration of 40 mg / ml and loaded on a 50 x 500 mm prepacked Chiral Technologies stainless steel column. The absorber was 20μ ChiralPak AD. Mobile phase is ethanol / triethylamine 100 / 0.1; The flow rate was 125 ml / min. The crude solution (25 mL) was loaded on the column every 12 minutes. A stationary recycle technique was used. Solvent was removed using a rotary evaporator. The final product was isolated as a gold oil, which solidified on standing; 399.0 g (96.4% recovery).

(2S, 5Z) -7-acetimidoylamino-2-amino-2-methyl-hept-5-enoic acid methyl ester, dihydrochloride hydrate

Example LL-12)

To a solution of the product of Example LL-11 (114.5 g, 0.425 mol) in methanol (2.4 L) under solids dibenzoyl-L-tartaric acid (152.5 g, 0.425 mol) and 88% formic acid (147 mL, 3.428 mol) ) Was added. A slurry of 5 wt% palladium (37.9 g) on calcium carbonate weakened with lead acetate, lead acetate in methanol (200 mL) was prepared under nitrogen. Then a solution of starting material was added to the light gray catalyst slurry under ambient temperature, followed by the methanol rinse (200 mL). The heterogeneous reaction mixture was heated at 45 ° C. for 1 hour 30 minutes. The onset of sustained gas evolution was found at about 40 ° C., indicating that the reaction is in progress. The mixture was cooled in an ice / water bath and then filtered through a plug of Supercell HyFlo. The yellow solution was concentrated in vacuo to give a viscous oil, which was dissolved between 2N aqueous HCl (2 L) and ethyl acetate (0.8 L) and layered. The layers were separated and the aqueous layer was washed once with ethyl acetate (0.8 L). Solvent and volatiles were removed under vacuum (= 70 ° C.) under vacuum. The intermediate product was used in the next step without further purification or characterization. LC-MS [M + H] ⁺ = 228.

Example LL)

The crude product of Example LL-12 (170 g) was dissolved in 2N aqueous HCl (1 L). The resulting orange solution was refluxed overnight, then cooled back to ambient temperature. The reaction mixture was concentrated to about one third of its volume and the acidic solution was passed through a solid phase extraction cartridge (25 g of C18 silica) to remove color and other impurities. The solvent was removed in vacuo (= 70 ° C.) to afford 208 g of crude product as a yellow gum.

The crude gum (31.3 g) was taken out of water (250 mL) and the material loaded onto a pretreated ion exchange column packed with acidic resin Dowex 50WX4-400 (about 600 g). The resin was first washed with water (1 L) followed by dilute aqueous HCl (1 L with 10/90 v / v HCl / water). The resin was eluted from the product with high ionic strength aqueous HCl (1.5 L of 20/90 v / v to 25/75 v / v concentration HCl / water). The aqueous solvent was removed in vacuo (= 70 ° C.) and the rubbery residue was taken out in 4% by volume aqueous trifluoroacetic acid (100 mL). The aqueous solvent was removed in vacuo (= 70 ° C.) and the procedure repeated one or more times. The residue was then dried under high vacuum to afford 32.2 g of gum as trifluoroacetic acid salt.

Crude (2S, 5Z) -7-acetimidoylamino-2-amino-2-methyl-hept-5-enoic acid, ditrifluoroacetic acid salt hydrate (32.2 g) was purified by reverse phase preparative chromatography. . It was dissolved in crude 0.1% aqueous TFA (50 mL) and loaded onto a 2-inch ID × 1 m stainless steel column packed with adsorbent (BHK polarity W / S, 50 □, 1.16 kg). The product was eluted at a flow rate of 120 ml / min with a step gradient from 0.1% aqueous TFA to 25/75 / 0.1 acetonitrile / water / TFA. The loading ratio was 36: 1 w / w silica to sample. The material was converted to HCl salt by removing the solvent in vacuo and repeatedly rinsing in vacuo with dilute aqueous HCl and solvent. Drying under high vacuum yielded 27.4 g of the title dihydrochloride hydrate as a yellow gum.

Example MM

(2R, 5Z) -2-Amino-2-methyl-7-[(1-iminoethyl) amino] -5-heptenoic acid, dihydrochloride

The isolated R-enantiomer during the separation described in Example LL-11 (1.13 g, 4.2 mmol) was dissolved in 11 ml of 25% aqueous acetic acid and heated to 60 ° C. Then, zinc powder (1.10 g) was added in equal portions four times at 30 minute intervals. After heating for a total of 3 hours, an aliquot was removed and checked by LC-MS, indicating that only unreacted starting material remained with the desired product in small amounts. The mixture was cooled to rt, filtered and stripped under vacuum to afford 2.31 g of a slushy white solid. The methyl ester was hydrolyzed with dilute hot HCl to afford the title compound. After purification by reverse phase chromatography and lyophilization, 0.31 g of title compound was obtained as a glassy solid.

Example NN

2S-amino-6-[(1-iminoethyl) amino] -N- (1H-tetrazol-5-yl) hexaneamide, hydrate, dihydrochloride

NN-1

Boc-L-Lys (Cbz) -OH (5 g, 13.18 mmol), 5-aminotetrazol monohydrate (1.36 g, 13.18 mmol) and N, N-diisopropylethyl in 20 mL dimethylformamide (DMF) To a stirred solution of amine (DIPEA) (5.1 g, 6.9 ml, 39.54 mmol) benzotriazol-1-yl-oxy-tris- (dimethylamino) phosphonium hexafluorophosphate (BOP) (6.4 g) at ambient temperature 14.49 mmol) was added.

After stirring for 1 hour, the reaction mixture was concentrated in vacuo. The residue was partitioned between 60 ml of ethyl acetate (EtOAc) and 50 ml of water. The layers were separated. The organic layer was washed with 50 ml of 1 M KHSO ₄ solution and twice with 50 ml of water. The product began to precipitate and the suspension was concentrated in vacuo to yield 9 g of crude compound. After drying, the product was purified by boiling in methylene chloride and then filtered to give 3.7 g of 1A (62.7%). The compound was characterized by ¹ H NMR.

NN-2

(2 g, 4.5 mmol) was reduced using Pd black in 50% EtOH / AcOH solution at 5 psi for 12 hours to yield 1.55 g (100%) of NN-2. The compound was characterized by ¹ H NMR.

NN-3

Triethylamine (TEA) (1.26 g, 1.74 mL, 12.45 mmol) was added to a stirred solution of NN-2 (1.55 g, 4.15 mmol) and methyl acetamidate hydrochloride (0.91 g, 8.31 mmol) in 25 mL DMF. I was. After stirring for 16 h under ambient temperature, the reaction mixture was filtered from triethylamine hydrochloride and the filtrate was concentrated under vacuum. The residue was dissolved in 50% AcOH and lyophilized. The crude product (2 g) was purified using reverse phase chromatography on a C-18 column to yield 0.9 g (52.3%) of 1C. The product was characterized by ¹ H NMR.

NN-4

(0.9 g, 2.17 mmol) was dissolved in 30 mL of acetic acid and 3 mL of 4N HCl / dioxane was added. The reaction was stirred for 20 minutes. Then 150 ml of ethyl ether was added under ambient temperature. After 2 hours, the precipitate was filtered off, washed with ethyl ether and dried to give 0.78 g of 1 (96%).

Example NN is a more potent i-NOS inhibitor than 2S-amino-6-[(1-iminoethyl) amino] hexaneamide (NIL amide) or NIL dimethylamide, or Example 1 is also more selective. Example NN is a good crystalline product as in all its intermediates. NIL, on the other hand, is glass, which makes it difficult to manipulate.

c. Biological data

Some or all of the following tests are used to demonstrate nitric oxide synthase inhibitor activity as well as useful pharmacological properties of the compounds of the present invention.

Citrulline assay of nitric oxide synthase

Nitric oxide synthase (NOS) activity can be measured by monitoring the conversion of L- [2,3- ³ H] arginine to L- [2,3- ³ H] citrulline (Bredt and Snyder See Snyder et al., Proc. Natl. Acad. Sci. USA, 87, 682-685, 1990 and Moore et al., J. Med. Chem. 39, 669-672, 1996. ). Human inducible NOS (hiNOS), human endothelial NOS (hecNOS) and human neurostructural NOS (hncNOS), respectively, are cloned from RNA extracted from human tissue. CDNA for human induced NOS (hiNOS) is isolated from a λcDNA library made of RNA extracted from colon samples from ulcerative colitis patients. CDNA for human endothelial NOS (hecNOS) is isolated from a λcDNA library made of RNA extracted from human embryonic venous endothelial cells (HUVEC), and cDNA for neurostructural NOS (hncNOS) is RNA extracted from the human cerebellum taken from the body. Isolate from the prepared λcDNA library. Recombinant enzymes are expressed in Sf9 insect cells using baculovirus vectors (Rodi et al., The Biology of Nitric Oxide, Pt. 4: Enzymology, Biochemistry and Immunology; Moncada, S., Feelisch, M. , Busse, R., Higgs, E., Eds .; Portland Press Ltd .: London, 1995; pp 447-450 ". Enzyme activity is isolated from soluble cell extracts and partially purified by DEAE-Sepharose chromatography. To measure NOS activity, 10 μl of enzyme was added to 40 μl of 50 mM Tris (pH 7.6) with or without test compound, 50 mM Tris (pH 7.6), bovine serum albumin 2.0 mg / ml, 2.0 mM DTT, 4.0 Reaction by adding 50 μl of reaction mixture containing mM CaCl ₂ , 20 μM FAD, 100 μM tetrahydrobiopterin, 0.4 mM NADPH and 0.9 μCi-containing 60 μM L-arginine containing L- [2,3- ³ H] -arginine Initiate. The final concentration of L-arginine in the assay is 30 μM. For hecNOS or hncNOS, calmodulin is included at a final concentration of 40-100 nM. After incubation at 37 ° C. for 15 minutes, 400 μl of a suspension of DOWEX 50W X-8 cation exchange resin (1 part resin, 3 parts buffer) in suspension buffer containing 10 mM EGTA, 100 mM HEPES, pH 5.5 and 1 mM L-citrulline Add to terminate the reaction. After mixing, the resin was allowed to settle and L- [2,3- ³ H] -citrulline formation was measured by counting an aliquot of the supernatant containing the liquid scintillation counter. The results are reported in Table I as IC ₅₀ values of the compounds for hiNOS, hecNOS and hncNOS.

RAW Cell Nitrite Assay

RAW 264.7 cells can be plated with confluency on 96-well tissue culture plates cultured overnight in the presence of LPS to induce NOS. One row of three to six wells can be left untreated to serve as a control for subtracting the nonspecific background. The medium can be removed well from each well and cells can be washed twice with Kreb-Ringers-Hepes (25 mM, pH 7.4) containing 2 mg / ml glucose. Cells were then placed on ice and incubated for 1 hour with 50 μl of buffer containing L-arginine (30 μM) +/− inhibitors. The assay can be initiated by warming the plate to 37 ° C. in a water bath for 1 hour. The production of nitrite by intracellular iNOS will be linear with time. To terminate the cell assay, cell plates were placed on ice, nitrite-containing buffer was removed and analyzed for nitrite using fluorescence assays for previously published nitrites. See TP Misko et al., Analytical Biochemistry , 214, 11-16 (1993).

Human Cartilage In Vitro Assay

Rinse the bone pieces twice with Dulbecco's phosphate buffered saline (GibcoBRL), rinse once with Dulbecco's Modified Eagles Medium (GibcoBRL), and no phenol red minimum essential medium (MEM) ( In a Petri dish containing GibcoBRL). Cartilage is cut into small in vitro cultures at approximately 15-45 mg weight and one or two in vitro cultures are placed in 96 or 48 well culture plates with 200-500 μl culture medium per well. The culture medium was prepared without L-arginine, without L-glutamine and without phenol red, usually modified minimum essential medium or with L-arginine, without insulin, without ascorbic acid, without L-glutamine And the normally modified serum member Neuman and Tytell (GibcoBRL) prepared without phenol red. All of these were 100 μM L-arginine (Sigma, 2 mM L-glutamine, 1 × HL-1 supplement (BioWhittacker), 50 mg / ml ascorbic acid (Sigma) and 150 pg / ml recombinant human IL- Supplemented prior to use of 1 □ (RD Systems) to induce nitric oxide synthase, compound is then added in 10 μl aliquots and in vitro cultures at 37 ° C. with 5% CO ₂ for 18-24 hours. The supernatant after one day is then removed, replaced with fresh culture medium containing recombinant human IL-1β and compound and incubated for another 20 to 24 hours.The supernatant is analyzed for nitrite by fluorescence assay ( Misko et al. , " Anal. Biochem. , 214, 11-16, 1993") All samples are repeated four times Unstimulated controls were cultured in medium in the absence of recombinant human IL-1β. IC ₅₀ values (Table 1) show nitrites with six different concentrations of inhibitors. Determined by plotting percent inhibition of production.

Table I gives examples of biological activities for some of the compounds of the present invention.

In vivo assay

Rats may be injected intraperitoneally with 1 to 12.5 mg / kg of endotoxin (LPS) without oral administration or administration of a nitric oxide synthase inhibitor. Plasma nitrite / nitrate levels can be measured 5 hours after treatment. The results can be used to indicate that administration of nitric oxide synthase inhibitors reduces the rise in plasma nitrite / nitrate levels, a reliable indicator of the production of nitric oxide induced by endotoxin. As shown in Table II, Example A ((2S, 5E) -2-Amino-6-fluoro-7-[(1-iminoethyl) amino] -5-heptenoic acid, dihydrochloride) was 0.1 It inhibits the increase in LPS-induced levels of plasma nitrite / nitrate with an ED ₅₀ observation of less than mg / kg, demonstrating its ability to inhibit inducible nitric oxide synthase activity in vivo.

Test on Time-Dependent Suppression

Compounds were evaluated for time dependent inhibition of human NOS isoforms by preincubating the compounds with enzymes at 37 ° C. in the presence of citrulline enzyme assay component, negative L-arginine for 0 to 60 minutes. Aliquots (10 μl) were withdrawn at 0, 10, 21 and 60 minutes to immediately contain L- [2,3- ³ H] -arginine and 30 μM final concentration of L-arginine at a final volume of 100 μl. Citrulline assay enzyme is added to the reaction mixture. The reaction proceeds at 37 ° C. for 15 minutes, terminated by addition of stop buffer, and chromatographed with the DOWEX 50W X-8 cation exchange ion exchange resin described for the citrulline NOS assay. The percent inhibition of NOS activity by the inhibitor was used as the activity inhibition rate compared to the control enzyme pre-cultured for the same time in the absence of the inhibitor. The data presented in Table III below are% inhibition after 21 and 60 minutes of preincubation of the inhibitor with enzymes.

In vitro study of airway endothelial cells

Immunocytochemistry for Bronchial Brushing: Endothelial cells obtained through bronchial brushing from healthy non-asthmatic individuals were prepared using Shandon Cytospin 3 centrifuges. The slides were air dried and fixed in ice cold acetone for 10 minutes. Endogenous peroxide activity was quenched by immersing the slides for 1 hour in 1% (v / v) H ₂ O ₂ in PBS containing 0.02% (w / v) sodium azide. Slides were washed three consecutive times in PBS for 5 minutes. Slides were incubated with 5% (v / v) normal pig serum for 20 minutes to prevent nonspecific binding. Anti-iNOS antibody (rabbit polyclonal affinity purified anti human iNOS MoAb 720; Pharmacia Corporation, St. Louis, MO) or pre-immune rabbit serum was then applied to the slides at a dilution of 1:50 for 1 hour at room temperature It was. The slides were then washed, incubated with biotinylated pig anti-rabbit lgG (1: 200) for 30 minutes at room temperature and washed. The slides were then incubated in avidin-horse radish peroxide (1: 500) for an additional 30 minutes. The slides were washed again and the antigens visualized with diaminobenzidine. Slides were counterstained with hematoxylin for 10 seconds, dehydrated in synergistic alcohol, cleared with xylene, and mounted using DPX. The cells were then observed under a light microscope.

Human Primary Airway Endothelial Cell Culture: Human primary airway endothelial cells are cultured as described above (Donnelly, LE) and Barnes, PJ (J. Am. J. Respir Cell Mol. Biol 24). : 295-303 (2000) ", the stable oxidative product of NO in cell culture medium, and the amount of nitrite are described in the spectral fluorometer method of Misco et al.," Anal. Biochem. 4: 11-16 (1993). It measured using the modified method.

Western blotting of iNOS from cultured endothelial cells: Endocrine sac was incubated with cytokine mixtures alone or with varying concentrations of L-NIL, followed by endothelial sac 0.25 mM ethylene diaminotetraacetic acid, petylmethylsulfonyl fluoride Lysing was performed in 50 mM Tris / HCl, pH 7.4, containing mM, 5 μg / ml antipain, 5 μg / ml lupetin and 5 μg / ml benzamidine. Protein concentration was measured using the BioRad protein assay kit according to the manufacturer's instructions. Cell proteins were purified by sodium dodecyl sulfate polyacrylamide gel electrophoresis sample buffer (0.0625 mM Tris / HCl, pH 6.8, 10% (v / v) glycerol, 1% (w / v) SDS, 1% (w / v) solubilized in β-mercaptoethanol and 0.01% (w / v) bromophenol blue). Protein (15 μg per lane) was dissolved by electrophoresis in 3-8% (w / v) tris-acetate SDS-polyacrylamide gels, and Hybond-Enhanced Chemiluminescene (ECL) nitro Moved to the cellulose membrane. The same protein load was determined by staining the blot with 0.1% (w / v) Ponceau S in 5% (v / v) acetic acid. Then 0.5M Tris at pH 7.4 containing nitro-cellulose 3% (w / v) normal goat serum, 1% (w / v) bovine serum albumin and 0.05% (v / v) Tween-20 Blocked overnight at 4 ° C. in HCl. Blots were washed in PBS containing 0.05% (v / v) Tween-20 and incubated for 1 hour in the presence of anti-human iNOS primary antibody (1: 1000). The blot was washed extensively and then incubated for 1 hour with anti-rabbit IgG conjugated with horseradish peroxidase (1: 4000). The blot was again washed extensively and visualized using the band as an ECL reagent.

Effect of L-NIL on iNOS Activity and Expression in Human Primary Airway Endothelial Cells: Blushing cells from normal individuals have been shown to express iNOS. The protein was distributed evenly throughout the cytoplasm of the basal cells, but was placed neatly under the cilia in columnar ciliated cells. INOS protein was induced by treatment of airway endothelial cells with IL-1β 50 ng / ml, TNF-α and IFN-γ (cytomix) for 24 hours, and from 2.20 ± 0.2 μM in the absence of cytomix In the presence of 3.5 ± 0.4 μM the accumulation of nitrite in the cell culture medium increased.

The effect of L-NIL on iNOS activity and expression in human primary airway endothelial cells is shown in FIGS. 1 and 2. The medium was harvested and the nitrite content was measured (FIG. 1). Cellular proteins were lysed on 3-8% tris-acetate polyacrylamide gels and immunoblotted against iNOS protein (FIG. 2).

1 shows that the increase in nitrite in the medium is inhibited depending on the amount used by adding L-NIL to the cell medium (IC ₅₀ approximately 5.7 μM). Figure 2 shows that the reduction of nitrite production is not mediated by inhibition of iNOS protein expression since the inclusion of L-NIL did not affect iNOS expression detected by immunoblot. Rather, it is expected to be the result of inhibition of enzyme activity.

Clinical research

A single center study was conducted at the National Heart and Lung Institute (NHLI) Clinical Laboratory, London, UK. The clinical protocol was submitted and recognized by the Ethics Committee of the Royal Brompton Hospital Trust, and this study was conducted according to Good Clinical practice criteria. Each subject gave written informed consent.

A 24 year old healthy adult volunteer and 24 mild asthmatic patients participated in this study. All subjects were nonsmokers for life. Asthmatic patients (n = 24) were mildly asthmatic patients according to the American Thoracic Society, and medication was inhaled only as essential. Patients had a compulsory exhalation rate (FEV ₁ ),> 15% reversibility with short-acting β ₂ -antagonist at 1 second and greater than 70%, and 20% of FEV ₁ (PC ₂₀ ) below 80 mg / ml It had an induced concentration of methacholine or histamine causing a decrease. In addition, skin prick tests positive for one or more conventional inhaled allergens (Dermatophagoides pteronyssinus, mixed grass pollen or cat's hair) were recorded for asthma patients, and none developed asthma or respiratory tract infections within four weeks of testing. All asthmatic patients had baseline exhalation NO levels above 15 ppb. Healthy control subjects (n = 24) matched age and gender and had no clinically serious disease. Healthy subjects were required to have baseline exhalation NO levels of 4 to 9 ppb.

The study included two panels of healthy volunteers and asthma patients receiving a single dose of oral compound (NN) or placebo in a double blind randomized manner. The study was carried out in two steps depending on the dose of compound (NN) under investigation. One panel consisted of mild to moderate asthma (n = 12) and healthy subjects (n = 12) receiving a single dose of 20 mg of compound (NN) or placebo and mild to moderate asthma (n = 12) and a second panel of healthy subjects (n = 12) received a single dose of 200 mg of compound (NN) and placebo. No patients or subjects participated in both panels of this study. Evaluation of the first panel was completed before moving to the second panel. The study evaluated the resistance and pharmacokinetics and monitoring levels of exhaled NO of orally administered compound (NN).

Determination of exhaled NO: Exhaled NO was measured using a chemiluminescent analyzer (Rogan 2000, Rochester, UK) and a method according to the International Guidelines. The analyzer was calibrated daily using 50-200 ppb of certified NO mixtures (BOC Special Gas, Guildford, UK) and ambient air NO readings on an hourly basis. Subjects were asked to refrain from vigorous exercise before all measurements. Each subject was seated for at least five minutes before all measurements were taken and continued to sit for the entire process. Subjects began to exhale at the volume of the residue and then inhaled rapidly at full lung capacity. Inhaled quickly, inhaled less than 2.5 seconds in healthy subjects and less than 4 seconds in asthmatic patients. After the analyzer became zero to NO, the subject was attached to the chemiluminescent analyzer at a constant stable flow rate of 250 ml / sec for low resistance (5-20 cm H ₂ O) from the total lung capacity over 15-20 seconds. Exhaled into the teflon tube of the hole. Sampling was performed at 250 ml / min. The NO peak response was interpreted as having a duration of more than 10 minutes. Two records were recorded continuously to calculate the average value.

Statistical Methods for Clinical Data: Exhaled NO data, including change from baseline and% change, were tested for normality using the Shapiro-Wilks test. Large deviations were found and non-parametric statistics were used. Changes from baseline were tested using the Wilcoxon signed rank test. The difference between the area under the curve (AUC), activity and placebo for change from baseline and% change was calculated. For normality tests, large deviations were found and non-parametric statistics were used. The significance of the total change was tested by comparing AUC with zero using the Wilcoxon signed rank test. The differences between treatments in each panel were tested using non-parametric analysis of crossover studies. The difference between the two active doses (20 mg and 200 mg) in the two panels was tested using the Wilcoxon rank test.

Forced exhalation volume (FEV ₁ ) and heart rate data for 1 second showed no significant deviation from normality using the Shapiro-Wilx test, therefore using parametric statistical methods and the significance of the change from baseline in the paired t-rank Test using the assay.

Effect of iNOS Inhibition in Normal Subjects and Asthma Patients: Exhaled NO levels in normal subjects and asthma patients after placebo and administration of Compound NN at a 20 or 200 mg dose are shown in FIG. 3. Changes in nitric oxide (NO) levels in exhalation were compared to placebo (△) compared to mild to moderate asthmatic patients (▲) and placebo (○). 20 mg) and (B) after oral administration of compound NN (200 mg). Vertical arrows mean the time points at which compound NN or placebo is administered. Mean values were given (n = 12).

Figure 3 shows that the change in AUC in exhaled NO did not differ significantly from zero for placebo in all four subjects. After administering Compound NN at two doses, there was a rapid decrease in exhaled NO, which was very significantly different in both healthy volunteers and asthma patients over the 72 hour evaluation. In addition, the difference in AUC change in NO for subjects who received 200 mg of compound NN was significantly different in subjects who received 20 mg of compound NN in both healthy volunteers (p <0.001) and asthma patients (p = <0.01). Higher than in AUC.

4 depicts the effect of oral administration of compound NN, blood pressure and heart rate on FEV ₁ . (A) shows the FEV ₁ after oral administration of compound NN (200 mg) in healthy subjects (●) as compared to mild to moderate asthma patients (▲) and placebo (○) compared to placebo (Δ). It is a change. (B) shows changes in systolic blood pressure after oral administration of compound NN (200 mg) (■) compared to placebo (□), and after oral administration of compound NN (200 mg) (◆) compared to placebo (◇). Diastolic blood pressure is a change. (C) is a change in heart rate after oral administration of 200 mg of compound NN (▲) as compared to placebo (Δ). Vertical arrows indicate the time points at which each dose of Compound NN or placebo is administered. Mean values were presented (n = 12).

4 shows that Compound NN was resistant without significant effects on heart rate, blood pressure and FEV ₁ . It also did not affect hematology or blood biochemistry.

The selective iNOS inhibitor compound NN was resistant in all subjects and resulted in a sharp decrease in exhaled NO after oral administration in both healthy volunteers and asthmatic patients. In addition, the decrease in exhaled NO persisted for 3 days in all groups. There were no significant changes in pulmonary function, blood pressure and heart rate, or significant changes in clinical hematology and biochemical parameters. Studies by intravenous administration of non-selective inhibitors of L-NMMA, NOS resulted in hypertension in both animals and humans. Description of hypertension in mutant mice lacking the gene for eNOS (Huang, PL et al., " Nature 377: 239-42 (1995)") suggest that L-NMMA causes hypertension through inhibition of eNOS. do. Thus, the good resistance of compound NN suggests that the compound significantly inhibits eNOS at test doses and encourages the study of iNOS inhibitors in asthma as well as other inflammatory diseases.

The long-term effect in reducing exhalation NO after a single dose suggests that the dosing regimen for the compound may be adjusted to allow for a single daily dose. Dosages greater than 200 mg may be over-maximal doses in the flat portion of the dose response curve. In addition, a dose of 200 mg induces 95% inhibition of exhaled NO in asthma, which is a high dose of non-specific inhibitors such as L-NMMA (Yates, DH et al., Am JRespir Crit Care Med 152: 892-896 (1995) ") and L-NAME (Gomez, FP et al." Eur Respir J 12: 856-871 (1998) ") as well as more selective amino-guanidines (Yates DH et al." Am J Greater than about 70% inhibition obtained from Respir Crit Care Med 154 "247-250 (1996)." Residual exhalation of less than 1 ppb in healthy volunteers and asthma patients, assuming complete inhibition of iNOS is selectively obtained. NO may be produced by structural nNOS and eNOS as well as by exogenous ambient sources.

Extended studies support an important role for NO in human asthma patients. Selective iNOS inhibitors include reduced pulmonary chemokine expression (Trifilieff, A. et al., J Immunol 165: 1526-33 (2000)) and allergic airway inflammation suppressed in mice with iNOS deficiency (Xiong, Y. et al. In rodent models of allergic airway inflammation associated with " The Journal of Immunology 162: 445-52 (1999)", it is known to inhibit eosinophil infiltration into the lungs (Koarai, A. et al ., Pulm. Pharmacol Ther. 13: 267-275 (2000) "). NO gas may be detected in increased amounts in exhalation from asthmatic patients (eg, Stirling, RG et al.," Thorax 53: 1030-34 (1998) ") peroxynit Wright (Sadeghi-Hashjin, G et al ., " Clin Exp Allergy 28: 1464-76 (1998)") increased in bronchial biopsies from asthmatic patients (Saleh, D. et al., TASEB J 12: 929-37 (1998) ") also, nitro tyrosine (et Hanazawa, T expiratory condensate." Am J Respir Crit Care Med 162: 1273-76 (2000) in ") and asthma Destroyed was increased in lung and airway of the patient (et Kaminsky, DA "J Allergy clin Immunol 104: 747-54 (2000)"). Recently, S- nitroso-thiols show a ventilation signal response to hypoxia, S-nitrosoglutathione is known to form when nitric oxide synthase (NOS) is activated in nerves and other tissues (Lipton, AJ et al., Nature 413: 171-74 (2001)).

Therapeutic evidence also exists for using selective iNOS therapy in chronic obstructive pulmonary disease (COPD) (see, eg, Barnes, PJ et al., N Engl J Med 343: 269-80 (2000)). Nitrotyrosine and iNOS have been increased in sputum cells (Ichinose, M. et al., Am J Respir Crit Care Med 162: 801-6 (2000)), but the use of exhaled NO as a practical marker for COPD is controversial. There is room (Maziak, W. et al., Am J Respir Crit Care Med 157: 998-1002 (1998), and Corradi, M. et al., [In Process Citation], Thorax 54: 572-75 (1999). ) And Rutgers, S. R et al., " Thorax 54: 576-80 (1999)". There is a possibility that NO is consumed by reactive oxidant species, which explains the fact that NO is reduced in severely stable COPD patients (Clini, E. et al., Thorax 53: 881-3 (1998) "). NO inhalation is also used for the treatment of chronic COPD (Ashutos, K. et al., Thorax 55: 109-13 (2000)).

c. Dosage, Formulation, and Route of Administration

Many of the iNOS selective inhibitor compounds useful in the present invention may have two or more asymmetric carbon atoms, and therefore may include racemates and stereoisomers such as diastereomers and enantiomers in their pure form or mixture. Such stereoisomers may be prepared using conventional techniques by reacting enantiomeric starting materials or separating isomers of the compounds of the invention. Isomers may include geometric isomers such as cis-isomers or trans-isomers across double bonds. All such isomers are contemplated among the compounds useful in the methods of the invention. The method also contemplates the use of tautomers, salts, solvates and prodrugs of iNOS selective inhibitor compounds.

For the methods of the invention, suitable routes of administration of selective iNOS inhibitors include any means of providing contact of these compounds with the site of action of the patient's body, such as the trachea, bronchus and lungs of the trachea. More specifically, suitable routes of administration include inhalation, intranasal mucosal administration, oral, intravenous, subcutaneous, rectal, topical, buccal (sublingual), intramuscular and intradermal administration, including oral or nasal inhalation.

For the prevention or treatment of diseases including respiratory diseases and disorders, asthma diseases, COPD including chronic bronchitis and pneumoconiosis, and cystic fibrosis, as well as other respiratory or pulmonary disorders with airway or lung inflammation, the method itself INOS selective inhibitors as or as pharmaceutically acceptable salts thereof. The term "pharmaceutically acceptable salts" includes salts commonly used to form alkali metal salts and to form addition salts of free acids or free bases. Salts are pharmaceutically acceptable salts, although their nature is not critical. Pharmaceutically acceptable salts are particularly useful as products of the process of the invention because they are more water soluble than the corresponding parent compound or neutral compound. Such salts should have a pharmaceutically acceptable anion or cation. Suitable pharmaceutically acceptable acid addition salts of the present invention can be prepared from inorganic acids or organic acids. Examples of such inorganic acids are hydrochloric acid, hydrobromic acid, hydroiodic acid, nitric acid, carbonic acid, sulfuric acid and phosphoric acid. Suitable organic acids are aliphatic, cycloaliphatic, aromatic, araliphatic, heterocyclic, carboxylic and sulfone organic acids such as formic acid, acetic acid, propionic acid, succinic acid, glycolic acid, gluconic acid, lactic acid, malic acid, tartaric acid, Citric acid, ascorbic acid, gluconic acid, maleic acid, fumaric acid, pyruvic acid, aspartic acid, glutamic acid, benzoic acid, anthranilic acid, mesylic acid, salicylic acid, p-hydrobenzoic acid, phenylacetic acid, mandelic acid, embonic acid (famoic acid), methanesulfuric acid Phenolic acid, ethylsulfonic acid, benzenesulfonic acid, sulfanilic acid, stearic acid, cyclohexylaminosulfonic acid, alzenic acid, galacturonic acid. Suitable pharmaceutically acceptable base addition salts of the compounds of the invention include metal salts from aluminum, calcium, lithium, magnesium, potassium, sodium and zinc, or N, N'-dibenzylethylenediamine, choline, chloroprop Organic salts prepared from caine, diethanolamine, ethylenediamine, meglumine (N-methylglucamine) and procaine. Suitable pharmaceutically acceptable acid addition salts of the compounds of the invention may, if possible, contain inorganic acids, such as hydrochloric acid, hydrobromic acid, hydrofluoric acid, boric acid, boric acid, phosphoric acid, metaphosphoric acid, nitric acid, carbonic acid (carbonates and hydrogen carbonate anions). And sulfonic acids and sulfuric acid, and organic acids such as acetic acid, benzenesulfonic acid, benzoic acid, citric acid, ethanesulfonic acid, fumaric acid, gluconic acid, glycolic acid, isoionic acid, lactic acid, lactobionic acid, maleic acid, malic acid And those derived from methanesulfonic acid, trifluoromethanesulfonic acid, succinic acid, toluenesulfonic acid, tartaric acid and trifluoroacetic acid. Chloride salts are particularly suitable for medical use. Suitable pharmaceutically acceptable base salts include ammonium salts, alkali metal salts such as sodium and potassium salts, and alkaline earth metal salts such as magnesium and calcium salts. All of these salts can be prepared by conventional means from the corresponding conjugated bases or conjugated acids of the compounds of the present invention, respectively, by reacting the appropriate acids or bases with the conjugated bases or conjugated acids of the compounds of the present invention.

In one embodiment, the iNOS selective inhibitors useful in the methods of the invention may be present as an acceptable carrier in the form of a pharmaceutical combination. The carrier should be acceptable and not deleterious to the patient in that it is compatible with the other ingredients of the pharmaceutical combination. Suitable forms for the carrier include both solids or liquids, and in exemplary embodiments the carrier is combined with the therapeutic compound as a tablet containing a unit-dosing combination, such as from about 0.05 to about 95 weight percent of the active compound. In other embodiments, other pharmacologically active ingredients are also present, including other compounds of the present invention. The pharmaceutical compounds of the present invention are prepared by any well known pharmaceutical technique consisting essentially of a mixture of ingredients.

Preferred unit dosage formulations are those containing one or more therapeutic compounds of the effective dosages, or combinations of the appropriate fractions, described herein below.

In general, the total daily dose of the iNOS selective inhibitor is about 0.001 mg / kg body weight / day to about 2500 mg / kg body weight / day. The dosage range for adult humans is generally from about 0.005 mg to about 10 g per day. Tablets or other forms of formulations provided in separate units may conveniently contain such a dosage or an amount of therapeutic compound effective for a plurality of the same dosage. For example, the selective iNOS inhibitor compound used in the present invention may be provided in units containing 5 to 500 mg, typically approximately 10 to about 200 mg.

In the case of pharmaceutically acceptable salts of the therapeutic compounds, the weights indicated above refer to the weight of the acid or base equivalents of the therapeutic compound derived from the salt.

For the methods described herein, the amount of selective iNOS inhibitor compound required to achieve the desired biological effect is determined by the particular individual compound (s) selected, the particular use, route of administration, clinical condition of the patient, and age, weight, gender and It depends on a number of factors, including diet.

The daily dosages described above for the various therapeutic compounds are administered in a single dose or multiple subdoses. Subdoses are administered twice to six times daily. In one embodiment, the dosage is administered in sustained release form effective to achieve the desired biological effect.

Delivery by inhalation, whether oral or nasal inhalation according to the method of the present invention, may be formulations that can be misted for delivery by inhalation, as is well known in the art. Metered dose inhalers or nebulizers provide aerosol delivery. Both devices can provide particle delivery in the particle size range, including particles in the preferred range of about 1 micron to about 5 microns. Particles larger than about 10 microns are primarily precipitated in the oral cavity and oropharyngeal, and particles smaller than about 0.5 microns are inhaled into the alveoli and do not deposit significantly in the lungs. Another device for inhalant therapy is a dry powder inhaler for delivering the therapeutic compound, for example using lactose or glucose powder. For all forms of inhalant therapy, factors other than particle size and device type depend on the amount of deposition in the lungs, single breath volume, respiratory rate and respiratory arrest. Thus, individuals who are instructed to inhale therapy according to the method of the present invention should be instructed to take a deep breath and stop each breath for a few seconds, preferably for about 5 to 10 seconds. Typically, the total daily dose of a therapeutic compound according to the method of the present invention is 1 to 4 puffs based on bid-qid (2 / day, 3 / day or 4 times / day), If necessary, it will be administered alone on a required basis.

Oral delivery according to the methods of the invention may include agents that provide delayed or sustained delivery of the drug to the respiratory system by any number of mechanisms, as is well known in the art. These include pH-sensitive release from dosage forms based on changes in pH of the small intestine, slow erosion of tablets or capsules, delay in the stomach depending on the properties of the preparation, bioadhesion of dosage forms into the mucous membrane of the intestinal tract, or activity from dosage forms. Including but not limited to, enzymatic release of drugs.

Oral delivery according to the methods of the present invention can be accomplished using solid, semisolid or liquid dosage forms. Suitable semisolid and liquid forms include, for example, syrups or liquids contained in gel capsules.

In order to practice the methods of the invention, pharmaceutical compositions suitable for oral administration may be presented in discrete units such as capsules, cachets, lozenges, or tablets, each of which contains a predetermined amount of one or more of the therapeutic compounds useful in the methods of the invention. As); As a powder or granules; As a suspension or solution in an aqueous or non-aqueous liquid; Or as oil-in-water or water-in-oil emulsions.

d. Example of embodiment

The following non-limiting examples are provided to illustrate various pharmaceutical compositions suitable for practicing the methods of treatment of the present invention.

Example 1 Pharmaceutical Composition

100 mg of tablets of the compositions disclosed in Table IV below can be prepared for oral administration using wet granulation techniques.

Example 2 Pharmaceutical Composition

100 mg of tablets of the compositions disclosed in Table V below can be prepared using a direct compression technique.

The embodiments described herein can be carried out by replacing the therapeutic compounds or inactive ingredients described collectively or specifically with those used in the previous examples.

The description and examples set forth herein are intended to enable those skilled in the art to familiarize themselves with the principles and practice of the present invention. Those skilled in the art can modify and apply the present invention in many forms and may best suit the requirements of a particular application. Accordingly, certain embodiments of the invention as disclosed are not intended to be exhaustive or to limit the invention.

Claims (38)

A method of treating, preventing or inhibiting a respiratory disease or disorder in a patient in need of treatment, prevention or inhibition of a respiratory disease or disorder, A compound of formula I, a compound of formula II, a compound of formula III, a compound of formula IV, a compound of formula V, a compound of formula VI, a compound of formula VII, a compound of formula VIII, A compound of formula (IX), a compound of formula (X), a compound of formula (XI), a compound of formula (XII), a compound of formula (XIII), a compound of formula (XIV), a compound of formula (XV), a PPA250 compound, and a pharmaceutically thereof A method comprising administering to a patient an effective amount of an inducible nitric oxide synthase selective inhibitor selected from the group consisting of an acceptable salt and a prodrug. Formula I Where R ¹ is selected from the group consisting of H, halo, and alkyl which may be optionally substituted with one or more halo, R ² is selected from the group consisting of H, halo, and alkyl which may be optionally substituted with one or more halo, At least one of R ¹ and R ² contains halo, R ⁷ is selected from the group consisting of H and hydroxy, J is selected from the group consisting of hydroxy, alkoxy and NR ³ R ⁴ , Wherein R ³ is selected from the group consisting of H, lower alkyl, lower alkylenyl and lower alkynyl, R ⁴ is selected from the group consisting of H, and a heterocyclic ring wherein at least one of the rings is carbon and 1 to about 4 heteroatoms are independently selected from oxygen, nitrogen, and sulfur, wherein the heterocyclic ring is heteroarylamino , N-aryl-N-alkylamino, N-heteroarylamino-N-alkylamino, haloalkylthio, alkanoyloxy, alkoxy, heteroaralkoxy, cycloalkoxy, cycloalkenyloxy, hydroxy, amino, thio, Nitro, lower alkylamino, alkylthio, alkylthioalkyl, arylamino, aralkylamino, arylthio, alkylsulfinyl, alkylsulfonyl, alkylsulfonamido, alkylaminosulfonyl, amidosulfonyl, monoalkyl amidosulfonyl , Dialkyl amidosulfonyl, monoarylamidosulfonyl, arylsulfonamido, diarylamidosulfonyl, monoalkyl monoaryl amidosulfonyl, arylsulfinyl, arylsulfonyl, Teraroarylthio, heteroarylsulfinyl, heteroarylsulfonyl, alkanoyl, alkenoyl, aroyl, heteroaroyl, aralkanoyl, heteroarkanoyl, haloalkanoyl, alkyl, alkenyl, alkynyl, alkylene Dioxy, haloalkylenedioxy, cycloalkyl, cycloalkenyl, lower cycloalkylalkyl, lower cycloalkenylalkyl, halo, haloalkyl, haloalkoxy, hydroxyhaloalkyl, hydroxyaralkyl, hydroxyalkyl, Hydroxyheteroaralkyl, haloalkoxyalkyl, aryl, aralkyl, aryloxy, aralkoxy, aryloxyalkyl, saturated heterocyclyl, partially saturated heterocyclyl, heteroaryl, heteroaryloxy, heteroaryloxyalkyl, arylalkyl , Heteroarylalkyl, arylalkenyl, heteroarylalkenyl, cyanoalkyl, dicyanoalkyl, carboxamidoalkyl, dicarboxamidoalkyl, cyanocarboalkoxy Kil, carboalkoxyalkyl, dicaralkoxyalkyl, cyanocycloalkyl, dicyanocycloalkyl, carboxamidocycloalkyl, dicarboxamidocycloalkyl, carboalkoxycyanocycloalkyl, carboalkoxycycloalkyl, di Carboalkoxycycloalkyl, formylalkyl, acylalkyl, dialkoxyphosphonoalkyl, dialkoxyphosphonoalkyl, phosphonoalkyl, dialkoxyphosphonoalkoxy, dialkoxyphosphonoalkoxy, phosphonoalkoxy, dialkoxyphosphonoalkyl Optionally substituted with amino, dialkoxyphosphonoalkylamino, phosphonoalkylamino, dialkoxyphosphonoalkyl, dialkylalkylphosphonoalkyl, guanidino, amidino or acylamino. Formula II Where X is selected from the group consisting of -S-, -S (O)-and -S (O) ₂ , R ¹² is C ₁ -C ₆ alkyl, C ₂ -C ₆ alkenyl, C ₂ -C ₆ alkynyl, C ₁ -C ₅ alkoxy-C ₁ alkyl and C ₁ -C ₅ alkylthio-C ₁ alkyl, wherein Each of these groups is optionally substituted with one or more substituents selected from the group consisting of -OH-, alkoxy and halogen; R ¹⁸ is selected from the group consisting of —OR ²⁴ and —N (R ²⁵ ) (R ²⁶ ), and R ¹³ is —H, —OH, —C (O) —R ²⁷ , —C (O) —OR ²⁸ And -C (O) -SR ²⁹ , or R ¹⁸ is -N (R ³⁰ )-, R ¹³ is -C (O)-, and R ¹⁸ and R ¹³ together with the atoms to which they are attached form a ring, or R ¹⁸ is -O-, R ¹³ is -C (R ³¹ ) (R ³² )-, wherein R ¹⁸ and R ¹³ together with the atoms attached thereto form a ring, When R ¹³ is -C (R ³¹ ) (R ³² )-, R ¹⁴ is -C (O) -OR ³³ , otherwise R ¹⁴ is -H, R ¹¹ , R ¹⁵ , R ¹⁶ and R ¹⁷ are independently —H, halogen, C ₁ -C ₆ alkyl, C ₂ -C ₆ alkenyl, C ₂ -C ₆ alkynyl and C ₁ -C ₅ alkoxy- Selected from the group consisting of C ₁ alkyl, R ¹⁹ and R ²⁰ are independently selected from the group consisting of -H, C ₁ -C ₆ alkyl, C ₂ -C ₆ alkenyl, C ₂ -C ₆ alkynyl and C ₁ -C ₅ alkoxy-C ₁ alkyl , R ²¹ is selected from the group consisting of -H, -OH, -C (O) -OR ³⁴ and -C (O) -SR ³⁵ , and R ²² is -H, -OH, -C (O) -OR ³⁶ And -C (O) -SR ³⁷ , or R ²¹ is -O-, R ²² is -C (O)-, and R ²¹ and R ²² together with the atoms to which they are attached form a ring, or R ²¹ is -C (O)-, R ²² is -O-, and R ²¹ and R ²² together with the atoms to which they are attached form a ring, R ²³ is C ₁ alkyl, R ²⁴ is selected from the group consisting of —H and C ₁ -C ₆ alkyl, and when R ²⁴ is C ₁ -C ₆ alkyl, R ²⁴ is selected from the group consisting of cycloalkyl, heterocyclyl, aryl and heteroaryl Optionally substituted with one or more residues, R ²⁵ is selected from the group consisting of -H, alkyl and alkoxy, R ²⁶ is -H, -OH, alkyl, alkoxy, -C (O) -R ³⁸ , -C (O) -OR ³⁹ and -C ( O) -SR ⁴⁰ , wherein when R ²⁵ and R ²⁶ are independently alkyl or alkoxy, R ²⁵ and R ²⁶ are independently selected from the group consisting of cycloalkyl, heterocyclyl, aryl and heteroaryl Optionally substituted with one or more residues, or R ²⁵ is -H and R ²⁶ is selected from the group consisting of cycloalkyl, heterocyclyl, aryl and heteroaryl, R ²⁷ , R ²⁸ , R ²⁹ , R ³⁰ , R ³¹ , R ³² , R ³³ , R ³⁴ , R ³⁵ , R ³⁶ , R ³⁷ , R ³⁸ , R ³⁹ and R ⁴⁰ are independently composed of -H and alkyl Selected from the group wherein the alkyl is optionally substituted with one or more residues selected from the group consisting of cycloalkyl, heterocyclyl, aryl and heteroaryl, Any R ¹¹ , R ¹² , R ¹³ , R ¹⁴ , R ¹⁵ , R ¹⁶ , R ¹⁷ , R ¹⁸ , R ¹⁹ , R ²⁰ , R ²¹ , R ²² , R ²³ , R ²⁴ , R ²⁵ , R ²⁶ , R ²⁷ , R ²⁸ , R ²⁹ , R ³⁰ , R ³¹ , R ³² , R ³³ , R ³⁴ , R ³⁵ , R ³⁶ , R ³⁷ , R ³⁸ , R ³⁹ and R ⁴⁰ are independently alkyl, alkenyl, alkyne When the residue is selected from the group consisting of one, alkoxy, alkylthio, cycloalkyl, heterocyclyl, aryl and heteroaryl, the residue is optionally substituted with one or more substituents selected from the group consisting of -OH, alkoxy and halogen . Formula III Where R ⁴¹ is H or methyl and R ⁴² is H or methyl. Formula IV Formula V Where R ⁴³ is hydrogen, halo, C ₁ -C ₅ alkyl, and C ₁ -C ₅ alkyl substituted with alkoxy or one or more halo, R ⁴⁴ is hydrogen, halo, C ₁ -C ₅ alkyl, and C ₁ -C ₅ alkyl substituted with alkoxy or one or more halo, R ⁴⁵ is C ₁ -C ₅ alkyl, or C ₁ -C ₅ alkyl substituted with alkoxy or one or more halo. Formula VI Wherein R ⁴⁶ is C ₁ -C ₅ alkyl wherein said C ₁ -C ₅ alkyl is optionally substituted with halo or alkoxy and said alkoxy is optionally substituted with one or more halo. Formula VII Where R ⁴⁷ is hydrogen, halo, C ₁ -C ₅ alkyl, and C ₁ -C ₅ alkyl substituted with alkoxy or one or more halo, R ⁴⁸ is hydrogen, halo, C ₁ -C ₅ alkyl, and C ₁ -C ₅ alkyl substituted with alkoxy or one or more halo, R ⁴⁹ is C ₁ -C ₅ alkyl, or C ₁ -C ₅ alkyl substituted with alkoxy or one or more halo. Formula VIII Where R ⁵⁰ is C ₁ -C ₅ alkyl, wherein the C ₁ -C ₅ alkyl is optionally substituted with halo or alkoxy and the alkoxy is optionally substituted with one or more halo. Formula IX Where R ⁵⁰ is selected from the group consisting of hydrogen, halo and C ₁ -C ₅ alkyl, wherein said C ₁ -C ₅ alkyl is optionally substituted with halo or alkoxy, said alkoxy is optionally substituted with one or more halo, R ⁵¹ is selected from the group consisting of hydrogen, halo and C ₁ -C ₅ alkyl, wherein said C ₁ -C ₅ alkyl is optionally substituted with halo or alkoxy, said alkoxy is optionally substituted with one or more halo, R ⁵² is C ₁ -C ₅ alkyl, wherein the C ₁ -C ₅ alkyl is optionally substituted with halo or alkoxy, the alkoxy is optionally substituted with one or more halo, R ⁵³ is selected from the group consisting of hydrogen, halo and C ₁ -C ₅ alkyl, wherein said C ₁ -C ₅ alkyl is optionally substituted with halo or alkoxy, said alkoxy is optionally substituted with one or more halo, R ⁵⁴ is selected from the group consisting of halo and C ₁ -C ₅ alkyl, wherein the C ₁ -C ₅ alkyl is optionally substituted with halo or alkoxy and the alkoxy is optionally substituted with one or more halo. Formula X Where R ⁵⁵ is C ₁ -C ₅ alkyl, wherein the C ₁ -C ₅ alkyl is optionally substituted with halo or alkoxy and the alkoxy is optionally substituted with one or more halo. Formula XI 2S-amino-6-[(1-iminoethyl) amino] -N- (1H-tetrazol-5-yl) hexaneamide, hydrate, dihydrochloride Formula XII Where R ⁷⁹ is selected from C _1-4 alkyl, C _3-4 cycloalkyl, C _1-4 hydroxyalkyl and C _1-4 haloalkyl. Formula XIII Formula XIV Formula XV In Formulas XIII, XIV, and XV, A is -R ⁵⁶ , -OR ⁵⁶ , C (O) N (R ⁵⁶ ) R ⁵⁷ , P (O) [N (R ⁵⁶ ) R ⁵⁷ ] ₂ , -N (R ⁵⁶ ) C (O) R ⁵⁷ , -N (R ⁷⁶ ) C (O) OR ⁵⁶ , -N (R ⁵⁶ ) R ⁷⁶ , -N (R ⁷¹ ) C (O) N (R ⁵⁶ ) R ⁷¹ , -S (O) _t R ⁵⁶ ,- SO ₂ NHC (O) R ⁵⁶ , -NHSO ₂ R ⁷⁷ , -SO ₂ NH (R ⁵⁶ ) H, -C (O) NHSO ₂ R ⁷⁷ and -CH = NOR ⁵⁶ , Each X, Y and Z is independently N or C (R ¹⁹ ), Each U is N or C (R ⁶⁰ ), where U is only N when X is N and Z and Y are CR ⁷⁴ , V is N (R ⁵⁹ ), S, O or C (R ⁵⁹ ) H, Each W is N or CH, Q is selected from the group consisting of a direct bond, -C (O)-, -O-, -C (= NR ⁵⁶ )-, S (O) _t and -N (R ⁶¹ )-, m is 0 or an integer from 1 to 4, n is 0 or an integer of 1 to 3, q is 0 or 1, r is 0 or 1, but when Q and V are heteroatoms, m, q and r cannot all be 0, A is -OR ⁵⁶ , N (R ⁵⁶ ) C (O) R ⁵⁷ , -N (R ⁷¹ ) C (O) OR ⁵⁷ , -N (R ⁵⁶ ) R ⁷⁶ , -N (R ⁷¹ ) C (O) When N (R ⁵⁶ ) R ⁷¹ , -S (O) _t R ⁵⁶ , where t is 0, or -NHSO ₂ R ⁷⁷ , all of n, q and r cannot be 0, and Q is hetero A and A are -OR ⁵⁶ , N (R ⁵⁶ ) C (O) R ⁵⁷ , -N (R ⁷¹ ) C (O) OR ⁵⁷ , -N (R ⁵⁶ ) R ⁷⁶ , N (R ⁷¹ ) C (O ) N (R ⁵⁶ ) R ⁷¹ , -S (O) _t R ⁵⁶ (where t is 0) or -NHSO ₂ R ⁷⁷ , m and n cannot both be 0, t is 0, 1 or 2, Is optionally substituted N-heterocyclyl, Is optionally substituted carbocyclyl or optionally substituted N-heterocyclyl, Each of R ⁵⁶ and R ⁵⁷ is hydrogen, optionally substituted C ₁ -C ₂₀ alkyl, optionally substituted cycloalkyl, — [C ₀ -C ₈ alkyl] -R ⁶⁴ ,-[C ₂ -C ₈ alkenyl ] -R ⁶⁴ ,-[C ₂ -C ₈ alkynyl] -R ⁶⁴ ,-[C ₂ -C ₈ alkyl] -R ⁶⁵ (optionally substituted with hydroxy),-[C ₁ -C ₈ ]- R ⁶⁶ (optionally substituted with hydroxy), independently selected from the group consisting of optionally substituted heterocyclyl, or R ⁵⁶ and R ⁵⁷ are N-heterocyclyl optionally substituted with a nitrogen atom attached to them, R ⁵⁸ is hydrogen, alkyl, cycloalkyl, optionally substituted aryl, haloalkyl,-[C ₁ -C ₈ alkyl] -C (O) N (R ⁵⁶ ) R ⁵⁷ ,-[C ₁ -C ₈ alkyl] -N (R ⁵⁶ ) R ⁵⁷ ,-[C ₁ -C ₈ alkyl] -R ⁶³ ,-[C ₂ -C ₈ alkyl] -R ⁶⁵ ,-[C ₁ -C ₈ alkyl] -R ⁶⁶ , and hetero or a direct bond with R ⁵⁸ - heterocyclyl selected from the group consisting of (halo, alkyl, alkoxy and imidazole optionally substituted with one or more substituents selected from the group consisting of thiazolyl) or, or Q is -N (R ⁵⁸⁾ R ⁵⁸ may further be aminocarbonyl, alkoxycarbonyl, alkylsulfonyl, monoalkylaminocarbonyl, dialkylaminocarbonyl and -C (= NR ⁷³ ) -NH ₂ , or -QR ⁵⁸ Together with -C (O) OH, -C (O) N (R ⁵⁶ ) R ⁵⁷ or Indicates, R ⁵⁹ is selected from the group consisting of hydrogen, alkyl, aryl, aralkyl and cycloalkyl, When A is —R ⁵⁶ or —OR ⁵⁶ , R ⁵⁹ may not be hydrogen; when V is CH, R ⁵⁹ may additionally be hydroxy, R ⁶⁰ is hydrogen, alkyl, aryl, aralkyl, haloalkyl, optionally substituted aralkyl, optionally substituted aryl, -OR ⁷¹ , -S (O) _t -R ⁷¹ , N (R ⁷¹ ) R ⁷⁶ , N (R ⁷¹ ) C (O) N (R ⁵⁶ ) R ⁷¹ , N (R ⁷¹ ) C (O) OR ⁷¹ , N (R ⁷¹ ) C (O) R ⁷¹ ,-[C ₀ -C ₈ alkyl] -C (H) [C (O) R ⁷¹ ] ₂ and-[C ₀ -C ₈ alkyl] -C (O) N (R ⁵⁶ ) R ⁷¹ , R ⁶¹ is hydrogen, alkyl, cycloalkyl,-[C ₁ -C ₈ alkyl] -R ⁶³ ,-[C ₂ -C ₈ ] alkyl] -R ⁶⁵ ,-[C ₁ -C ₈ alkyl] -R ⁶⁶ , Acyl, -C (O) R ⁶³ , -C (O)--[C ₁ -C ₈ alkyl] -R ⁶³ , alkoxycarbonyl, optionally substituted aryloxycarbonyl, optionally substituted aralkoxycarbonyl , Alkylsulfonyl, optionally substituted aryl, optionally substituted heterocyclyl, alkoxycarbonylalkyl, carboxyalkyl, optionally substituted arylsulfonyl, aminocarbonyl, monoalkylaminocarbonyl, dialkylaminocarbonyl , Optionally substituted arylaminocarbonyl, aminosulfonyl, monoalkylaminosulfonyl dialkylaminosulfonyl, arylaminosulfonyl, arylsulfonylaminocarbonyl, optionally substituted N-heterocyclyl, -C ( = NH) -N (CN) R ⁵⁶ , -C (O) R ⁷⁸ -N (R ⁵⁶ ) R ⁵⁷ , -C (O) -N (R ⁵⁶ ) R ⁷⁸ -C (O) OR ⁵⁶ Selected from Each of R ⁶³ and R ⁶⁴ is optionally substituted with one or more substituents selected from the group consisting of haloalkyl, cycloalkyl (optionally substituted with halo, cyano, alkyl or alkoxy), carbocyclyl (halo, alkyl and alkoxy) And heterocyclyl (optionally substituted with alkyl, aralkyl or alkoxy), and Each R ⁶⁵ is halo, alkoxy, optionally substituted aryloxy, optionally substituted aralkoxy, optionally substituted -S (O) _t -R ⁷⁷ , acylamino, amino, monoalkylamino, dialkylamino, Independently selected from the group consisting of (triphenylmethyl) amino, hydroxy, mercapto, alkylsulfonamido, Each R ⁶⁶ is independently selected from the group consisting of cyano, di (alkoxy) alkyl, carboxy, alkoxycarbonyl, aminocarbonyl, monoalkylaminocarbonyl and dialkylaminocarbonyl, Each of R ⁶⁷ , R ⁶⁸ , R ⁶⁹ , R ⁷⁰ , R ⁷² and R ⁷⁵ is independently hydrogen or alkyl, Each R ⁷¹ is independently hydrogen, alkyl, optionally substituted aryl, optionally substituted aralkyl or cycloalkyl, R ⁷³ is hydrogen, NO ₂ or toluenesulfonyl, Each R ⁷⁴ is independently hydrogen, alkyl (optionally substituted with hydroxy), cyclopropyl, halo or haloalkyl, Each R ⁷⁶ is independently hydrogen, alkyl, cycloalkyl, optionally substituted aryl, optionally substituted aralkyl, —C (O) R ⁷⁷ or —SO ₂ R ⁷⁷ , or R ⁷⁶ is N-heterocyclyl optionally substituted with R ⁵⁶ and the nitrogen attached thereto; or R ⁷⁶ is N-heterocyclyl optionally substituted with R ⁷¹ and nitrogen attached thereto, Each R ⁷⁷ is independently alkyl, cycloalkyl, optionally substituted aryl or optionally substituted aralkyl, R ⁷⁸ is an amino acid residue. The method of claim 1, Said respiratory disease or condition is selected from the group consisting of asthma disease and chronic obstructive pulmonary disease (COPD). The method of claim 1, The respiratory disease or condition may be allergen-induced asthma, exercise-induced asthma, contaminant-induced asthma, cold-induced asthma, virus-induced asthma, chronic bronchitis with normal air flow, chronic bronchitis, pneumoconiosis , Asthma bronchitis, bullous disease, cystic fibrosis, pigeon fancier's disease, peasant lung, acute respiratory distress syndrome, pneumonia, aspiration or inhalation injury, fat embolism in the lungs, acidosis inflammation of the lungs, acute pulmonary edema, Acute altitude sickness, post cardiac surgery, acute pulmonary hypertension, neonatal permanent pulmonary hypertension, prenatal respiratory syndrome, vitreous membrane disease, acute pulmonary embolism, heparin-protamine reaction, sepsis, asthma persistence and hypoxia. The method of claim 1, Said respiratory disease is an asthma disease. The method of claim 4, wherein Said asthma disease is allergen-induced asthma. The method of claim 4, wherein The asthma disease is contaminant-induced asthma. The method of claim 4, wherein Wherein said asthma disease is exercise-induced asthma. The method of claim 4, wherein Wherein said asthma disease is virus-induced asthma. The method of claim 4, wherein Wherein said asthma disease is cold-induced asthma. The method of claim 1, The respiratory disease is chronic obstructive pulmonary disease (COPD). The method of claim 1, And said respiratory disease is pneumoconiosis. The method of claim 1, Said respiratory disease is bronchitis. The method of claim 12, Said respiratory disease is chronic bronchitis with normal air flow. The method of claim 12, Said respiratory disease is chronically impaired bronchitis. The method of claim 1, Said respiratory disease is asthma bronchitis. The method of claim 1, Said respiratory disease is a bullous disease. The method of claim 1, The respiratory disease is cystic fibrosis. The method of claim 1, Said respiratory disease is bronchiectasis. The method of claim 1, Wherein said inducible nitric oxide synthase inhibitor is a compound of formula (I) or a pharmaceutically acceptable salt thereof. Formula I Where R ¹ is selected from the group consisting of H, halo, and alkyl which may be optionally substituted with one or more halo, R ² is selected from the group consisting of H, halo, and alkyl which may be optionally substituted with one or more halo, At least one of R ¹ and R ² contains halo, R ⁷ is selected from the group consisting of H and hydroxy, J is selected from the group consisting of hydroxy, alkoxy and NR ³ R ⁴ , Wherein R ³ is selected from the group consisting of H, lower alkyl, lower alkylenyl and lower alkynyl, R ⁴ is selected from the group consisting of H, and a heterocyclic ring wherein at least one of the rings is carbon and from 1 to about 4 heteroatoms are independently selected from oxygen, nitrogen, and sulfur, wherein the heterocyclic ring is heteroarylamino , N-aryl-N-alkylamino, N-heteroarylamino-N-alkylamino, haloalkylthio, alkanoyloxy, alkoxy, heteroaralkoxy, cycloalkoxy, cycloalkenyloxy, hydroxy, amino, thio, Nitro, lower alkylamino, alkylthio, alkylthioalkyl, arylamino, aralkylamino, arylthio, alkylsulfinyl, alkylsulfonyl, alkylsulfonamido, alkylaminosulfonyl, amidosulfonyl, monoalkyl amidosulfonyl , Dialkyl amidosulfonyl, monoarylamidosulfonyl, arylsulfonamido, diarylamidosulfonyl, monoalkyl monoaryl amidosulfonyl, arylsulfinyl, arylsulfonyl, Teraroarylthio, heteroarylsulfinyl, heteroarylsulfonyl, alkanoyl, alkenoyl, aroyl, heteroaroyl, aralkanoyl, heteroarkanoyl, haloalkanoyl, alkyl, alkenyl, alkynyl, alkylene Dioxy, haloalkylenedioxy, cycloalkyl, cycloalkenyl, lower cycloalkylalkyl, lower cycloalkenylalkyl, halo, haloalkyl, haloalkoxy, hydroxyhaloalkyl, hydroxyaralkyl, hydroxyalkyl, Hydroxyheteroaralkyl, haloalkoxyalkyl, aryl, aralkyl, aryloxy, aralkoxy, aryloxyalkyl, saturated heterocyclyl, partially saturated heterocyclyl, heteroaryl, heteroaryloxy, heteroaryloxyalkyl, arylalkyl , Heteroarylalkyl, arylalkenyl, heteroarylalkenyl, cyanoalkyl, dicyanoalkyl, carboxamidoalkyl, dicarboxamidoalkyl, cyanocarboalkoxy Kil, carboalkoxyalkyl, dicaralkoxyalkyl, cyanocycloalkyl, dicyanocycloalkyl, carboxamidocycloalkyl, dicarboxamidocycloalkyl, carboalkoxycyanocycloalkyl, carboalkoxycycloalkyl, di Carboalkoxycycloalkyl, formylalkyl, acylalkyl, dialkoxyphosphonoalkyl, dialkoxyphosphonoalkyl, phosphonoalkyl, dialkoxyphosphonoalkoxy, dialkoxyphosphonoalkoxy, phosphonoalkoxy, dialkoxyphosphonoalkyl Optionally substituted with amino, dialkoxyphosphonoalkylamino, phosphonoalkylamino, dialkoxyphosphonoalkyl, dialkylalkylphosphonoalkyl, guanidino, amidino or acylamino. The method of claim 19, And said inducible nitric oxide synthase inhibitor is selected from the group consisting of the following compounds or pharmaceutically acceptable salts thereof. (2S, 5E) -2-amino-6-fluoro-7-[(1-iminoethyl) amino] -5-heptenoic acid, dihydrochloride, monohydrate; (2S, 5E / Z) -2-Amino-6-fluoro-7-[(1-iminoethyl) amino] -5-heptenoic acid, dihydrochloride; (2S, 5Z) -2-Amino-6-fluoro-7-[(1-iminoethyl) amino] -5-heptenoic acid, dihydrochloride; (2S, 5Z) -2-Amino-6-fluoro-7-[(1-iminoethyl) amino] -5-heptenoic acid, trihydrochloride, dihydrate; (2R, 5E) -2-Amino-6-fluoro-7-[(1-iminoethyl) amino] -5-heptenoic acid, dihydrochloride, monohydrate; (2S, 5E) -2-amino-6-fluoro-7-[(1-iminoethyl) amino] -5-heptenoic acid, dihydrochloride, monohydrate; And (2S, 5E) -2-Amino-6-fluoro-7-[(1-hydroxyiminoethyl) amino] -5-heptenoic acid. The method of claim 1, The inducible nitric oxide synthase inhibitor (2S, 5E) -2-Amino-6-fluoro-7-[(1-iminoethyl) amino] -N- (1H-tetrazol-5-yl) -5-heptenamide, dihydrochloride, Or a pharmaceutically acceptable salt thereof. The method of claim 1, Wherein said inducible nitric oxide synthase inhibitor is a compound of formula (II): or a pharmaceutically acceptable salt thereof. Formula II Where X is selected from the group consisting of -S-, -S (O)-and -S (O) ₂ , R ¹² is C ₁ -C ₆ alkyl, C ₂ -C ₆ alkenyl, C ₂ -C ₆ alkynyl, C ₁ -C ₅ alkoxy-C ₁ alkyl and C ₁ -C ₅ alkylthio-C ₁ alkyl, wherein Each of these groups is optionally substituted with one or more substituents selected from the group consisting of -OH-, alkoxy and halogen; R ¹⁸ is selected from the group consisting of -OR ²⁴ and -N (R ²⁵ ) (R ²⁶ ), and R ¹³ is -H, -OH, -C (O) -R ²⁷ , -C (O) -OR ²⁸ And -C (O) -SR ²⁹ , or R ¹⁸ is -N (R ³⁰ )-, R ¹³ is -C (O)-, and R ¹⁸ and R ¹³ together with the atoms to which they are attached form a ring, or R ¹⁸ is -O-, R ¹³ is -C (R ³¹ ) (R ³² )-, wherein R ¹⁸ and R ¹³ together with the atoms attached thereto form a ring, When R ¹³ is -C (R ³¹ ) (R ³² )-, R ¹⁴ is -C (O) -OR ³³ , otherwise R ¹⁴ is -H, R ¹¹ , R ¹⁵ , R ¹⁶ and R ¹⁷ are independently —H, halogen, C ₁ -C ₆ alkyl, C ₂ -C ₆ alkenyl, C ₂ -C ₆ alkynyl and C ₁ -C ₅ alkoxy- Selected from the group consisting of C ₁ alkyl, R ¹⁹ and R ²⁰ are independently selected from the group consisting of -H, C ₁ -C ₆ alkyl, C ₂ -C ₆ alkenyl, C ₂ -C ₆ alkynyl and C ₁ -C ₅ alkoxy-C ₁ alkyl , R ²¹ is selected from the group consisting of -H, -OH, -C (O) -OR ³⁴ and -C (O) -SR ³⁵ , and R ²² is -H, -OH, -C (O) -OR ³⁶ And -C (O) -SR ³⁷ , or R ²¹ is -O-, R ²² is -C (O)-, and R ²¹ and R ²² together with the atoms to which they are attached form a ring, or R ²¹ is -C (O)-, R ²² is -O-, and R ²¹ and R ²² together with the atoms attached to them form a ring, R ²³ is C ₁ alkyl, R ²⁴ is selected from the group consisting of —H and C ₁ -C ₆ alkyl, and when R ²⁴ is C ₁ -C ₆ alkyl, R ²⁴ is selected from the group consisting of cycloalkyl, heterocyclyl, aryl and heteroaryl Optionally substituted with one or more residues, R ²⁵ is selected from the group consisting of -H, alkyl and alkoxy, R ²⁶ is -H, -OH, alkyl, alkoxy, -C (O) -R ³⁸ , -C (O) -OR ³⁹ and -C ( O) -SR ⁴⁰ , wherein when R ²⁵ and R ²⁶ are independently alkyl or alkoxy, R ²⁵ and R ²⁶ are independently selected from the group consisting of cycloalkyl, heterocyclyl, aryl and heteroaryl Optionally substituted with one or more residues, or R ²⁵ is -H and R ²⁶ is selected from the group consisting of cycloalkyl, heterocyclyl, aryl and heteroaryl, R ²⁷ , R ²⁸ , R ²⁹ , R ³⁰ , R ³¹ , R ³² , R ³³ , R ³⁴ , R ³⁵ , R ³⁶ , R ³⁷ , R ³⁸ , R ³⁹ and R ⁴⁰ are independently composed of -H and alkyl Selected from the group, said alkyl is optionally substituted with one or more residues selected from the group consisting of cycloalkyl, heterocyclyl, aryl and heteroaryl, Any R ¹¹ , R ¹² , R ¹³ , R ¹⁴ , R ¹⁵ , R ¹⁶ , R ¹⁷ , R ¹⁸ , R ¹⁹ , R ²⁰ , R ²¹ , R ²² , R ²³ , R ²⁴ , R ²⁵ , R ²⁶ , R ²⁷ , R ²⁸ , R ²⁹ , R ³⁰ , R ³¹ , R ³² , R ³³ , R ³⁴ , R ³⁵ , R ³⁶ , R ³⁷ , R ³⁸ , R ³⁹ and R ⁴⁰ are independently alkyl, alkenyl, alkyne When the residue is selected from the group consisting of one, alkoxy, alkylthio, cycloalkyl, heterocyclyl, aryl and heteroaryl, the residue is optionally substituted with one or more substituents selected from the group consisting of -OH, alkoxy and halogen . The method of claim 22, And said inducible nitric oxide synthase inhibitor is selected from the group consisting of the following compounds or pharmaceutically acceptable salts thereof. S- [2-[(1-iminoethyl) amino) ethyl] -2-methyl-L-cysteine; 2-[[[2-[(1-iminoethyl) amino] ethyl] thio] methyl] -O-methyl-D-serine, dihydrochloride; S-[(1R) -2-[(1-iminoethyl) amino] -1-methylethyl] -2-methyl-L-cysteine, dihydrochloride; S-[(1S) -2-[(1-iminoethyl) amino] -1-methylethyl] -2-methyl-L-cysteine, dihydrochloride; S- [2-[(1-iminoethyl) amino] ethyl] -2-ethyl-L-cysteine, dihydrochloride, 2-[[[[2- (1-iminoethyl) amino] ethyl] thio] methyl] -D-valine, dihydrochloride; S- [2- (1-Iminoethylamino) ethyl] -2-methyl- (D / L) -cysteine, bistrifluoroacetate; (2R) -2-amino-3 [[2-[(1-iminoethyl) amino] ethyl] sulfinyl] -2-methylpropanoic acid, dihydrochloride; And (2R) -2-amino-3 [[2- (1-iminoethyl) amino] ethyl] sulfonyl] -2-methylpropanoic acid dihydrochloride. The method of claim 1, Wherein said inducible nitric oxide synthase inhibitor is a compound of formula III: Formula III Wherein R ⁴¹ is H or methyl and R ⁴² is H or methyl. The method of claim 24, And said inducible nitric oxide synthase inhibitor is selected from the group consisting of the following compounds or pharmaceutically acceptable salts thereof. (2S, 5Z) -2-Amino-6-methyl-7-[(1-iminoethyl) amino] -5-heptenoic acid, dihydrochloride; (2S, 5E) -2-Amino-6-methyl-7-[(1-iminoethyl) amino] -5-heptenoic acid, dihydrochloride; (2S, 5Z) -2-Amino-7-[(1-iminoethyl) amino] -5-heptenoic acid, dihydrochloride; And (2S, 5E) -2-Amino-7-[(1-iminoethyl) amino] -5-heptenoic acid, dihydrochloride. The method of claim 1, Wherein said inducible nitric oxide synthase inhibitor is a compound of formula IV: or a pharmaceutically acceptable salt thereof. Formula IV The method of claim 26, And said inducible nitric oxide synthase inhibitor is selected from the group consisting of the following compounds or pharmaceutically acceptable salts thereof. (αR, 2S) -α-aminohexahydro-7-imino-1H-azepine-2-hexanoic acid, trihydrate, hydrochloride; (αS, 2R) -α-aminohexahydro-7-imino-1H-azepine-2-hexanoic acid, trihydrate, hydrochloride; (αS, 2S) -α-aminohexahydro-7-imino-1H-azepine-2-hexanoic acid, trihydrate, hydrochloride; (αR, 2S) -α-aminohexahydro-7-imino-1H-azepine-2-hexanoic acid, trihydrate, hydrochloride; (αS, 2S) -α-aminohexahydro-7-imino-1H-azepine-2-hexanoic acid, trihydrate, hydrochloride; (2S, 4Z) -2-Amino-6-[(2R) -hexahydro-7-imino-1H-azin-2-yl] -4-hexenoic acid; And (2S, 4E) -2-Amino-6-[(2R) -hexahydro-7-imino-1H-azin-2-yl] -4-hexenoic acid. The method of claim 1, Wherein said inducible nitric oxide synthase inhibitor is a compound of Formula (V): or a pharmaceutically acceptable salt thereof. Formula V Where R ⁴³ is hydrogen, halo, C ₁ -C ₅ alkyl, and C ₁ -C ₅ alkyl substituted with alkoxy or one or more halo, R ⁴⁴ is hydrogen, halo, C ₁ -C ₅ alkyl, and C ₁ -C ₅ alkyl substituted with alkoxy or one or more halo, R ⁴⁵ is C ₁ -C ₅ alkyl, or C ₁ -C ₅ alkyl substituted with alkoxy or one or more halo. The method of claim 28, Said inducible nitric oxide synthase inhibitor is selected from the group consisting of the following compounds or pharmaceutically acceptable salts thereof. (E) -2-amino-2-methyl-6-[(1-iminoethyl) amino] -4-hexenoic acid, dihydrochloride; (R, E) -2-amino-2-methyl-6-[(1-iminoethyl) amino] -4-hexenoic acid, dihydrochloride; And (S, E) -2-amino-2-methyl-6-[(1-iminoethyl) amino] -4-hexenoic acid, dihydrochloride. The method of claim 1, Wherein said inducible nitric oxide synthase inhibitor is a compound of Formula VI: or a pharmaceutically acceptable salt thereof. Formula VI Wherein R ⁴⁶ is C ₁ -C ₅ alkyl wherein said C ₁ -C ₅ alkyl is optionally substituted with halo or alkoxy and said alkoxy is optionally substituted with one or more halo. The method of claim 30, The inducible nitric oxide synthase inhibitor 2-amino-2-methyl-6-[(1-iminoethyl) amino] -4-hexenoic acid, dihydrochloride or a pharmaceutically acceptable salt thereof. The method of claim 1, Wherein said inducible nitric oxide synthase inhibitor is a compound of Formula (VII): or a pharmaceutically acceptable salt thereof. Formula VII Where R ⁴⁷ is hydrogen, halo, C ₁ -C ₅ alkyl, and C ₁ -C ₅ alkyl substituted with alkoxy or one or more halo, R ⁴⁸ is hydrogen, halo, C ₁ -C ₅ alkyl, and C ₁ -C ₅ alkyl substituted with alkoxy or one or more halo, R ⁴⁹ is C ₁ -C ₅ alkyl, or C ₁ -C ₅ alkyl substituted with alkoxy or one or more halo. The method of claim 32, And said inducible nitric oxide synthase inhibitor is selected from the group consisting of the following compounds or pharmaceutically acceptable salts thereof. (2R / S, 4Z) -2-Amino-2-methyl-7-[(1-iminoethyl) amino] -4-heptenoic acid, dihydrochloride; (2S, 5E) -2-Amino-2-methyl-6-fluoro-7-[(1-iminoethyl) amino] -5-heptenoic acid, dihydrochloride; (2S, 5E) -2-Amino-2-methyl-6-fluoro-7-[(1-iminoethyl) amino] -5-heptenoic acid, dihydrochloride; (2R, 5E) -2-Amino-2-methyl-6-fluoro-7-[(1-iminoethyl) amino] -5-heptenoic acid, dihydrochloride; (2R / S, 5E) -2-Amino-2-methyl-6-fluoro-7-[(1-iminoethyl) amino] -5-heptenoic acid, dihydrochloride; (2S, 5Z) -2-Amino-2-methyl-7-[(1-iminoethyl) amino] -5-heptenoic acid, dihydrochloride; And (2R, 5Z) -2-Amino-2-methyl-7-[(1-iminoethyl) amino] -5-heptenoic acid, dihydrochloride. The method of claim 1, Wherein said inducible nitric oxide synthase inhibitor is a compound of Formula (X): or a pharmaceutically acceptable salt thereof. Formula X Where R ⁵⁵ is C ₁ -C ₅ alkyl, wherein the C ₁ -C ₅ alkyl is optionally substituted with halo or alkoxy and the alkoxy is optionally substituted with one or more halo. The method of claim 1, Wherein said inducible nitric oxide synthase inhibitor is a compound of Formula (XII) or a pharmaceutically acceptable salt thereof. Formula XII Where R ⁷⁹ is selected from C _1-4 alkyl, C _3-4 cycloalkyl, C _1-4 hydroxyalkyl and C _1-4 haloalkyl. 36. The method of claim 35 wherein The inducible nitric oxide synthase inhibitor, S-((R) -2- (1-iminoethylamino) propyl) -L-cysteine, S-((S) -2- (1-iminoethylamino) propyl) -L-cysteine, S-((R / S) -2- (1-iminoethylamino) propyl) -L-cysteine, S-((R) -2- (1-iminoethylamino) propyl) -D-cysteine, S-((S) -2- (1-iminoethylamino) propyl) -D-cysteine, S-((R / S) -2- (1-iminoethylamino) propyl) -D-cysteine, S-((R / S) -2- (1-iminoethylamino) butyl) -L-cysteine, S-((R / S) -2- (1-iminoethylamino, 2-cyclopropyl) ethyl) -L-cysteine and S-((R / S) -2- (1-iminoethylamino, 3-hydroxy) propyl) -L-cysteine, Or a pharmaceutically acceptable salt thereof. The method of claim 1, Said inducible nitric oxide synthase inhibitor is selected from the group consisting of a compound of formula XIII, a compound of formula XIV, a compound of formula XV, and a pharmaceutically acceptable salt thereof. Formula XIII Formula XIV Formula XV In the above formulas, A is -R ⁵⁶ , -OR ⁵⁶ , C (O) N (R ⁵⁶ ) R ⁵⁷ , P (O) [N (R ⁵⁶ ) R ⁵⁷ ] ₂ , -N (R ⁵⁶ ) C (O) R ⁵⁷ , -N (R ⁷⁶ ) C (O) OR ⁵⁶ , -N (R ⁵⁶ ) R ⁷⁶ , -N (R ⁷¹ ) C (O) N (R ⁵⁶ ) R ⁷¹ , -S (O) _t R ⁵⁶ ,- SO ₂ NHC (O) R ⁵⁶ , -NHSO ₂ R ⁷⁷ , -SO ₂ NH (R ⁵⁶ ) H, -C (O) NHSO ₂ R ⁷⁷ and -CH = NOR ⁵⁶ , Each X, Y and Z is independently N or C (R ¹⁹ ), Each U is N or C (R ⁶⁰ ), where U is only N when X is N and Z and Y are CR ⁷⁴ , V is N (R ⁵⁹ ), S, O or C (R ⁵⁹ ) H, Each W is N or CH, Q is selected from the group consisting of a direct bond, -C (O)-, -O-, -C (= NR ⁵⁶ )-, S (O) _t and -N (R ⁶¹ )-, m is 0 or an integer from 1 to 4, n is 0 or an integer of 1 to 3, q is 0 or 1, r is 0 or 1, but when Q and V are heteroatoms, m, q and r cannot all be 0, A is -OR ⁵⁶ , N (R ⁵⁶ ) C (O) R ⁵⁷ , -N (R ⁷¹ ) C (O) OR ⁵⁷ , -N (R ⁵⁶ ) R ⁷⁶ , -N (R ⁷¹ ) C (O) When N (R ⁵⁶ ) R ⁷¹ , -S (O) _t R ⁵⁶ , where t is 0, or -NHSO ₂ R ⁷⁷ , all of n, q and r cannot be 0, and Q is hetero A and A are -OR ⁵⁶ , N (R ⁵⁶ ) C (O) R ⁵⁷ , -N (R ⁷¹ ) C (O) OR ⁵⁷ , -N (R ⁵⁶ ) R ⁷⁶ , N (R ⁷¹ ) C (O ) N (R ⁵⁶ ) R ⁷¹ , -S (O) _t R ⁵⁶ (where t is 0) or -NHSO ₂ R ⁷⁷ , m and n cannot both be 0, t is 0, 1 or 2, Is optionally substituted N-heterocyclyl, Is optionally substituted carbocyclyl or optionally substituted N-heterocyclyl, Each of R ⁵⁶ and R ⁵⁷ is hydrogen, optionally substituted C ₁ -C ₂₀ alkyl, optionally substituted cycloalkyl, — [C ₀ -C ₈ alkyl] -R ⁶⁴ ,-[C ₂ -C ₈ alkenyl ] -R ⁶⁴ ,-[C ₂ -C ₈ alkynyl] -R ⁶⁴ ,-[C ₂ -C ₈ alkyl] -R ⁶⁵ (optionally substituted with hydroxy),-[C ₁ -C ₈ ]- R ⁶⁶ (optionally substituted with hydroxy), independently selected from the group consisting of optionally substituted heterocyclyl, or R ⁵⁶ and R ⁵⁷ are N-heterocyclyl optionally substituted with a nitrogen atom attached to them, R ⁵⁸ is hydrogen, alkyl, cycloalkyl, optionally substituted aryl, haloalkyl,-[C ₁ -C ₈ alkyl] -C (O) N (R ⁵⁶ ) R ⁵⁷ ,-[C ₁ -C ₈ alkyl] -N (R ⁵⁶ ) R ⁵⁷ ,-[C ₁ -C ₈ alkyl] -R ⁶³ ,-[C ₂ -C ₈ alkyl] -R ⁶⁵ ,-[C ₁ -C ₈ alkyl] -R ⁶⁶ , and hetero or a direct bond with R ⁵⁸ - heterocyclyl selected from the group consisting of (halo, alkyl, alkoxy and imidazole optionally substituted with one or more substituents selected from the group consisting of thiazolyl) or, or Q is -N (R ⁵⁸⁾ R ⁵⁸ may further be aminocarbonyl, alkoxycarbonyl, alkylsulfonyl, monoalkylaminocarbonyl, dialkylaminocarbonyl and -C (= NR ⁷³ ) -NH ₂ , or -QR ⁵⁸ Together with -C (O) OH, -C (O) N (R ⁵⁶ ) R ⁵⁷ or Indicates, R ⁵⁹ is selected from the group consisting of hydrogen, alkyl, aryl, aralkyl and cycloalkyl, When A is —R ⁵⁶ or —OR ⁵⁶ , R ⁵⁹ may not be hydrogen; when V is CH, R ⁵⁹ may additionally be hydroxy, R ⁶⁰ is hydrogen, alkyl, aryl, aralkyl, haloalkyl, optionally substituted aralkyl, optionally substituted aryl, -OR ⁷¹ , -S (O) _t -R ⁷¹ , N (R ⁷¹ ) R ⁷⁶ , N (R ⁷¹ ) C (O) N (R ⁵⁶ ) R ⁷¹ , N (R ⁷¹ ) C (O) OR ⁷¹ , N (R ⁷¹ ) C (O) R ⁷¹ ,-[C ₀ -C ₈ alkyl] -C (H) [C (O) R ⁷¹ ] ₂ and-[C ₀ -C ₈ alkyl] -C (O) N (R ⁵⁶ ) R ⁷¹ , R ⁶¹ is hydrogen, alkyl, cycloalkyl,-[C ₁ -C ₈ alkyl] -R ⁶³ ,-[C ₂ -C ₈ ] alkyl] -R ⁶⁵ ,-[C ₁ -C ₈ alkyl] -R ⁶⁶ , Acyl, -C (O) R ⁶³ , -C (O)--[C ₁ -C ₈ alkyl] -R ⁶³ , alkoxycarbonyl, optionally substituted aryloxycarbonyl, optionally substituted aralkoxycarbonyl , Alkylsulfonyl, optionally substituted aryl, optionally substituted heterocyclyl, alkoxycarbonylalkyl, carboxyalkyl, optionally substituted arylsulfonyl, aminocarbonyl, monoalkylaminocarbonyl, dialkylaminocarbonyl , Optionally substituted arylaminocarbonyl, aminosulfonyl, monoalkylaminosulfonyl dialkylaminosulfonyl, arylaminosulfonyl, arylsulfonylaminocarbonyl, optionally substituted N-heterocyclyl, -C ( = NH) -N (CN) R ⁵⁶ , -C (O) R ⁷⁸ -N (R ⁵⁶ ) R ⁵⁷ , -C (O) -N (R ⁵⁶ ) R ⁷⁸ -C (O) OR ⁵⁶ Selected from Each of R ⁶³ and R ⁶⁴ is optionally substituted with one or more substituents selected from the group consisting of haloalkyl, cycloalkyl (optionally substituted with halo, cyano, alkyl or alkoxy), carbocyclyl (halo, alkyl and alkoxy) And heterocyclyl (optionally substituted with alkyl, aralkyl or alkoxy), and Each R ⁶⁵ is halo, alkoxy, optionally substituted aryloxy, optionally substituted aralkoxy, optionally substituted -S (O) _t -R ⁷⁷ , acylamino, amino, monoalkylamino, dialkylamino, Independently selected from the group consisting of (triphenylmethyl) amino, hydroxy, mercapto, alkylsulfonamido, Each R ⁶⁶ is independently selected from the group consisting of cyano, di (alkoxy) alkyl, carboxy, alkoxycarbonyl, aminocarbonyl, monoalkylaminocarbonyl and dialkylaminocarbonyl, Each of R ⁶⁷ , R ⁶⁸ , R ⁶⁹ , R ⁷⁰ , R ⁷² and R ⁷⁵ is independently hydrogen or alkyl, Each R ⁷¹ is independently hydrogen, alkyl, optionally substituted aryl, optionally substituted aralkyl or cycloalkyl, R ⁷³ is hydrogen, NO ₂ or toluenesulfonyl, Each R ⁷⁴ is independently hydrogen, alkyl (optionally substituted with hydroxy), cyclopropyl, halo or haloalkyl, Each R ⁷⁶ is independently hydrogen, alkyl, cycloalkyl, optionally substituted aryl, optionally substituted aralkyl, —C (O) R ⁷⁷ or —SO ₂ R ⁷⁷ , or R ⁷⁶ is N-heterocyclyl optionally substituted with R ⁵⁶ and the nitrogen attached thereto; or R ⁷⁶ is N-heterocyclyl optionally substituted with R ⁷¹ and nitrogen attached thereto, Each R ⁷⁷ is independently alkyl, cycloalkyl, optionally substituted aryl or optionally substituted aralkyl, R ⁷⁸ is an amino acid residue. The method of claim 1, Wherein said inducible nitric oxide synthase inhibitor is a PPA250 compound or a pharmaceutically acceptable salt thereof.